Conditions: Renal Transplant
Keywords: Renal Transplant Tacrolimus UNC Phase IV
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 97
Healthy volunteers: no

Brief summary:
Objective: Investigate the direct correlation of CYP3A5 genotype with tacrolimus trough levels and clinical outcomes. The primary endpoint of this study is to evaluate the proportion of patients reaching target levels (8-10 ng/mL) on Day 3 and Day 7 after kidney transplantation.

Detailed description:
Participants: All new kidney transplant recipients aged 18 to 65 years who are admitted at UNC-CH and provided informed consent will be included in this study (Unless they meet the exclusion criteria specified). A total of an anticipated 260 subjects will be included in the study, 130 of which will be included in the pharmacogenomic group and the remaining 130 will be in the control group.

Procedures (methods): The pharmacogenomic group will partake in a 12-month study comprising of two periods, Genotype-Guided Initial Dosing Intervention and Follow-up.

Briefly, patients on transplant waitlist will be screened for eligibility. At the pre-intervention assessment (Study Day 0), buccal swab samples for genotyping will be collected on all eligible patients who provided informed consent (performed in real time). Results of the genotyping test will be incorporated into electronic medical record (EMR). The initial tacrolimus dose will be based on genotype: 0.1 mg/kg/day (non- expressers) or 0.2 mg/kg/day, with maximum of 20 mg/day (expressers) given in 2 divided doses. Eligible patients who consented to receive genotype-guided tacrolimus dose will enter the pharmacogenomic group and will receive the initial tacrolimus dosing based on genotype results following kidney transplantation (Study Day 1). Subsequent tacrolimus dosing will then be adjusted according to trough concentrations (C0) and therapeutic target concentrations. The genotype-guided dosing recommendation for tacrolimus only refers to the initial tacrolimus dose. All patients in the pharmacogenomic group will be followed from Study Day 2 and up to 12 months to assess long-term outcome.

Age-, race-, and disease-matched patients who had previously received kidney transplantation with standard tacrolimus dosing from 2010 to present will also be asked to give consent for genotyping (historical controls). These patients will be included in the control group and their safety and efficacy data will be collected retrospectively for up to 12 months from the initiation of first tacrolimus dose.

As there are confounding variables, including age, race and disease state that may impact the results of the study, our study design incorporates an overall matching strategy, so that we can identify a well-matched control group. First, to control for differences in care over time, patients in the pharmacogenomic group will be matched to controls enrolled from 2010 to present. This time period was selected as there had been no major changes in standard of care or treatment regimen since 2010. After eligibility is met, control patients will be selected to match the pharmacogenomic group using a computerized matching algorithm that has been optimized to match baseline demographic and disease characteristics that have been identified a priori as likely to influence the treatment response to tacrolimus. To balance the trade-off between minimizing bias and maximizing matched sample size, a systematic approach will be conducted to identify the number of matched control patients for each patient in the pharmacogenomic group. This approach will include the following steps: 1) run the desired matching algorithm, starting with 1:1 (one control to one patient in the pharmacogenomic group) matching and iterating until the maximum desired number of potential controls per treated subject is reached; 2) for each iteration, test for covariate balance; and (3) generate numeric summaries and graphical plots of the balance statistics across all iterations in order to determine the optimal number.

The selection of patients for the control group using a matching algorithm will be conducted by an independent statistician in a blinded and unbiased manner. The statistician will have no knowledge of survival outcome, other outcome data, and genotype. The algorithm will not be used to guide treatment in any way.

Arms:
arm 1: Subjects in this treatment arm will receive initial tacrolimus based on their genotype i.e., CYP3A5\*1/\*1 and CYP3A5\*1/\*3 (Expressers) will receive the initial tacrolimus dose of 0.2 mg/kg/day, with maximum of 20 mg/day in 2 divided doses. For CYP3A5\*3/\*3 (Non-Expressers), the subjects will receive initial tacrolimus dose of 0.1 mg/kg/day in 2 divided doses. arm 2: Subjects in the prospective control group will receive standard tacrolimus dosing as recommended per package insert and will not be dosed based on their genotype. Similarly, subjects that underwent renal transplant after 2010 and received standard tacrolimus dosing (per package insert) will serve as historical controls.

Interventions: intervention 1: Tacrolimus
Intervention descriptions: intervention 1: See description in arm/group sections

Protocol document:
Prot_SAP_000.pdf: 
 
STUDY OF PHARMACOGENOMIC-GUIDED  
TACROLIMUS DOSING AND MONITORING IN KIDNEY  
TRANSPLANT RECIPIENTS 
 
 
 
NCT number NCT03020589 
Document Date 07/09/2018 
 


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CONFIDENTIAL 
CLINICAL RESEARCH PROTOCOL 
 
 
 
 
 
 
 
 
STUDY OF PHARMACOGENOMIC-GUIDED 
TACROLIMUS DOSING AND MONITORING IN KIDNEY 
TRANSPLANT RECIPIENTS 
 
 
 
 
 
 
Study Sponsor: 
UNC Eshelman School of Pharmacy  
Center for Pharmacogenomics and Individualized Therapy  
Genetic Medicine Building 
120 Mason Farm Road 
Chapel Hill, NC 27599 
 
Funding Sponsor: 
UNC Eshelman School of Pharmacy  
Center for Pharmacogenomics and Individualized Therapy  
Genetic Medicine Building 
120 Mason Farm Road 
Chapel Hill, NC 27599 
 
Study Product: 
Tacrolimus – Generic 
IRB Study Number: 
16-2073 
Study Phase: 
Postmarketing; Phase 4 
 
 
 
 
 
 
 
 
 
 
Initial version: 07OCT2016 
Second version: 14JUL2017 
Third version: 09JUL2018 
 
 


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Table of Contents 
 
1 
STUDY SUMMARY .............................................................................................................................. 1 
1 
ETHICAL CONSIDERATIONS ............................................................................................................ 1 
1.1 
SUBJECT INFORMATION AND CONSENT ............................................................................................. 1 
2 
INTRODUCTION .................................................................................................................................. 1 
2.1 
BACKGROUND AND RATIONALE ......................................................................................................... 2 
2.2 
PREVIOUS CLINICAL EXPERIENCE WITH GENOTYPE-GUIDED TACROLIMUS DOSING ............................. 3 
3 
STUDY OBJECTIVES ......................................................................................................................... 3 
3.1 
PRIMARY ENDPOINT......................................................................................................................... 4 
3.2 
SECONDARY ENDPOINTS ................................................................................................................. 4 
3.3 
EXPLORATORY ENDPOINTS .............................................................................................................. 4 
4 
STUDY DESIGN ................................................................................................................................... 4 
4.1 
GENERAL DESIGN ............................................................................................................................ 4 
4.2 
RATIONALE...................................................................................................................................... 6 
4.3 
PATIENT SELECTION ........................................................................................................................ 6 
4.4 
GENOTYPING AND DOSING ADMINISTRATION ..................................................................................... 7 
5 
SUBJECT SELECTION AND WITHDRAWAL .................................................................................... 8 
5.1 
INCLUSION CRITERIA ........................................................................................................................ 8 
5.2 
EXCLUSION CRITERIA ...................................................................................................................... 8 
5.3 
PATIENT RECRUITMENT AND SCREENING .......................................................................................... 8 
5.4 
REMOVAL OF PATIENTS FROM THE GENOTYPE-GUIDED TACROLIMUS THERAPY .................................. 8 
6 
STUDY DRUG ...................................................................................................................................... 9 
6.1 
DESCRIPTION .................................................................................................................................. 9 
6.2 
TREATMENT REGIMEN ..................................................................................................................... 9 
6.2.1 
Pharmacogenomic-guided dosing algorithm for the initial tacrolimus dose .......................... 9 
6.2.2 
Therapeutic Target Concentrations..................................................................................... 10 
7 
STUDY PROCEDURES ..................................................................................................................... 10 
7.1 
PRE-INTERVENTION ASSESSMENTS ................................................................................................ 10 
7.2 
PHARMACOGENOMIC INTERVENTION ............................................................................................... 11 
7.3 
POST-INTERVENTION OR FOLLOW-UP ASSESSMENTS ...................................................................... 11 
8 
STATISTICAL PLAN ........................................................................................................................... 2 
8.1 
GENERAL CONSIDERATIONS ............................................................................................................. 2 
8.2 
SAMPLE SIZE DETERMINATION ......................................................................................................... 2 
8.3 
STATISTICAL METHODS .................................................................................................................... 2 
8.4 
MATCHING STRATEGY...................................................................................................................... 3 
9 
SAFETY AND ADVERSE EVENTS ..................................................................................................... 3 
9.1 
DEFINITIONS .................................................................................................................................... 3 
9.2 
RECORDING OF ADVERSE EVENTS ................................................................................................... 4 
9.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS..................................... 5 
9.3.1 
Investigator reporting ............................................................................................................ 5 
9.4 
MEDICAL MONITORING ..................................................................................................................... 5 
9.5 
INTERIM ANALYSIS ........................................................................................................................... 5 
10 
DATA HANDLING AND RECORD KEEPING ..................................................................................... 6 


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10.1 
CONFIDENTIALITY ............................................................................................................................ 6 
10.2 
RECORDS RETENTION ..................................................................................................................... 6 
10.3 
METHOD OF DATA COLLECTION ........................................................................................................ 7 
10.4 
DATA OBTAINED FOR ANALYSIS ........................................................................................................ 7 
10.4.1 
Data to be collected .............................................................................................................. 7 
10.4.1.1 
Clinical and Environmental Factors ............................................................................... 7 
10.4.1.2 
Genetic Data ................................................................................................................. 7 
10.4.2 
Historical Data ....................................................................................................................... 7 
10.4.3 
Missing Data .......................................................................................................................... 8 
10.5 
DATA MANAGEMENT ........................................................................................................................ 8 
11 
STUDY MANAGEMENT ...................................................................................................................... 8 
11.1 
STUDY MONITORING PLAN ............................................................................................................... 8 
11.2 
CENTRAL LABORATORY COMPLIANCE ............................................................................................... 8 
11.3 
PATIENT ADHERENCE WITH STUDY MEDICATION ............................................................................... 8 
11.4 
DEVIATIONS FROM THE PROTOCOL ................................................................................................... 9 
11.5 
ADHERENCE TO THE PROTOCOL ....................................................................................................... 9 
11.6 
AMENDMENTS TO THE PROTOCOL .................................................................................................... 9 
11.7 
OBLIGATIONS OF INVESTIGATORS ..................................................................................................... 9 
12 
STUDY FINANCES .............................................................................................................................. 9 
12.1 
FUNDING SOURCE ........................................................................................................................... 9 
12.2 
CONFLICT OF INTEREST ................................................................................................................... 9 
12.3 
SUBJECT STIPENDS OR PAYMENTS ................................................................................................... 9 
13 
REFERENCES ..................................................................................................................................... 9 
 
 


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Protocol Revision History 
 
The following describes the revision history for this protocol. 
 
Protocol Version 
History of Changes 
Protocol Version 1 
Initial version approved by the IRB 
Protocol Version 2 
The following amendments to this protocol were made: 
• 
Tacrolimus dosing: To meet the current standard of care at 
UNC, the initial dosing of tacrolimus was changed from 0.3 to 
0.2 mg/kg/day for CYP3A5 expresser and from 0.15 
mg/kg/day to 0.1 mg/kg/day for CYP3A5 nonexpresser; 
tacrolimus is to be given in two divided doses with a maximum 
total daily dose of 20 mg/day. Safety data regarding the initial 
tacrolimus dosing of 0.1 mg/kg/day and 0.2 mg/kg/day are 
available in the drug’s package insert. 
• 
Safety monitoring: The statement “AEs will be collected 
following kidney transplantation” was added in Section 9.2 to 
clarify that AEs will be collected after subjects have received 
kidney transplantation and are eligible to receive tacrolimus. 
Protocol Version 3 
A sentence in the table of schedule of activities was added to clarify 
that assessment of drug-related AEs may be done in person or by 
phone. 


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List of Abbreviations 
 
The following abbreviations and specialized terms are used in this study protocol. 
 
Abbreviation or specialized 
term 
Explanation 
AE 
Adverse event 
ADR 
Adverse drug reaction 
C0 
Trough concentration 
CFR 
Code of Federal Regulations 
CGI 
Cancer Genetics Incorporated 
CI 
Confidence Intervals 
CL 
Central Laboratory 
CLIA 
Clinical Laboratory Improvement Amendment 
CrCl 
Creatinine Clearance 
eGFR 
Estimated Glomerular Filtration Rate 
CPIT 
Center for Pharmacogenomics and Individualized Therapy 
CRF 
Case Report Form 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
ITT 
Intent-to-treat 
PD 
Pharmacodynamics 
PHI 
Protected health information 
PID 
Participant identification number 
PK 
Pharmacokinetic 
REDCap 
Research Electronic Data Capture 
SAE 
Serious adverse event 
SOP 
Standard operating procedure 
SNP 
Single nucleotide polymorphism 
UNC-CH 
University of North Carolina in Chapel Hill 
 


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1 Study Summary 
Title 
Study of Pharmacogenomic-guided Tacrolimus Dosing and Monitoring in 
Kidney Transplant Recipients 
Short Title 
Pharmacogenomic-guided Dosing of Tacrolimus 
Protocol Number 
16-2073 
Phase 
Postmarketing; Phase 4 
Methodology 
This is a study of pharmacogenomic-guided tacrolimus dosing and drug 
monitoring in kidney transplant recipients. The pharmacogenomic group will 
partake in a 12-month study comprising of two periods, Genotype-Guided 
Initial Dosing Intervention and Follow-up.  
 
Briefly, patients currently on the waitlist to receive kidney transplantation at 
UNC-CH who are being seen by the multi-disciplinary kidney transplantation 
team will be screened for eligibility. At the pre-intervention assessment (Study 
Day 0), buccal swab samples for genotyping will be collected on all eligible 
patients who provided informed consent (performed in real time). All buccal 
swab samples collected will then be de-identified and sent to Cancer 
Genetics Incorporated (CGI) for CYP3A5 genotyping. Results of the 
genotyping test will be incorporated into electronic medical record (EMR) in 
addition to the genotype-guided dosing recommendation for each patient. 
The initial tacrolimus dose will be based on genotype: 0.1 mg/kg/day (non-
expressers) or 0.2 mg/kg/day (expressers) given in 2 divided doses; 
maximum total daily dose of tacrolimus is 20 mg/day. Eligible patients who 
consented to receive genotype-guided tacrolimus dose will enter the 
pharmacogenomic group and will receive the initial tacrolimus dosing based 
on genotype results following kidney transplantation (Study Day 1). 
Subsequent tacrolimus dosing will then be adjusted according to trough 
concentrations (C0) and therapeutic target concentrations of 8-10 ng/mL (0 to 
4 months), 6-8 ng/mL (5 to 12 months), and 5-7 ng/mL (>12 months). Dose 
adjustments and therapeutic drug monitoring will be conducted according to 
standard UNC-CH procedure. Hence, the genotype-guided dosing 
recommendation for tacrolimus only refers to the initial tacrolimus dose. All 
patients in the pharmacogenomic group will be followed from Study Day 2 
and up to 12 months from the initiation of the first tacrolimus dose to assess 
long-term outcome (Follow-up Period). Safety and efficacy measures will be 
evaluated throughout the study per UNC-CH kidney transplant procedure. 
 
Age-, race-, and disease-matched patients who had previously received 
kidney transplantation with standard tacrolimus dosing from 2010 to present 
will also be asked to give consent for genotyping (historical controls). These 
patients will be included in the control group and their safety and efficacy data 
will be collected retrospectively for up to 12 months from the initiation of first 
tacrolimus dose. 
Study Duration 
12-month follow up post-transplant 
Study Center(s) 
Single-center study 
Objectives 
Investigate the direct correlation of CYP3A5 genotype with tacrolimus trough 
levels and clinical outcomes. The primary endpoint of this study is to evaluate 
the proportion of patients reaching target levels (8-10 ng/ml) on Day 3 and 
Day 7 after kidney transplantation. 


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Number of Subjects 
260  
Inclusion/Exclusion 
Criteria 
Inclusion Criteria: All new kidney transplant recipients aged 18 to 65 years 
who are admitted at UNC-CH and provided informed consent will be included 
in this study (unless they meet exclusion criteria). 
 
Patients will be excluded from participating in the study to receive genotype-
guided tacrolimus dosing if he/she meets any of the exclusion criteria 
described below. 
• 
Recipients who did not consent to participate in the 
study. 
• 
Highly sensitized patients (ie, pretransplant T or B cell 
flow crossmatch positive) 
• 
Recipients of ABO incompatible kidney transplant 
• 
Recipients with preformed donor-specific antibodies 
(DSA) 
• 
HLA identical kidney transplant 
• 
Recipients of non-kidney transplant 
• 
Recipients of repeat transplant if they are on 
immunosuppression at the time of transplant 
• 
Patients using medications that have known 
pharmacokinetic (PK) drug interaction with tacrolimus 
• 
Patients in whom tacrolimus therapy is contraindicated 
Study Product, Dose, 
Route, Regimen 
All patients who consented will undergo genotype testing prior to transplant. 
Therapy for oral tacrolimus will be prescribed by the kidney transplant team 
as part of a standard immunosuppression agent given to kidney transplant 
recipients starting at 0.1 mg/kg/day (nonexpressers) or 0.2 mg/kg/day 
(expressers) given in 2 divided doses. The genotype-guided dosing only 
refers to the initial tacrolimus dose. Subsequent tacrolimus dosing will then 
be adjusted according to trough concentrations and desired therapeutic 
concentrations. 
Reference therapy 
Pharmacogenomic group is compared to age-, race-, and disease-matched 
patients who received standard dose of tacrolimus as part of their 
immunosuppression regimen from 2010 to present (historical controls). 
 
As the arrival of the donor kidney may come quickly and unexpectedly, it is 
possible that enrolled subjects may receive kidney transplantation prior to the 
incorporation of the CYP3A5 genotype result into EMR. In this case, patients 
will receive tacrolimus based on current standard tacrolimus dosing (0.1 
mg/kg/day given in two divided doses) and will be included in the control 
group (prospective controls). 
 
Hence, the control group will consist of both historical controls and potentially 
few prospective controls, if the scenario described above occurred. 


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Statistical 
Methodology 
All analyses will be performed on the basis of the ITT principle. Continuous 
variables will be summarized as medians with first to third quartiles. 
Appropriate conventional statistical analyses will be used to assess the 
primary and secondary endpoints. Categorical data will be compared using 
χ2 or Fisher’s exact test, as appropriate; some comparisons will require 
correction for potential confounders such as race/ethnicity; these will be 
performed using the Cochrane-Mantel-Haenszel test. Continuous variables 
will be compared using the Wilcoxon test or the t-test. The incidence of an 
adverse outcome (ie, graft rejection, graft loss, renal impairment, tacrolimus 
toxicities, and death) will be estimated using Kaplan–Meier curves. 
Depending on the distribution of the data, time-to-event (ie, time to reach 
therapeutic target range) analysis will be performed using an appropriate 
parametric, nonparametric, or semi-parametric survival analysis method. 
 


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1 Ethical Considerations 
This is a clinical study protocol for a human research study. This study is to be conducted according to 
United States (US) and international standards of Good Clinical Practice (FDA Title 21 part 312 and 
International Conference on Harmonization [ICH] guidelines), applicable government regulations, and 
Institutional research policies and procedures grounded on the principles found within the Declaration of 
Helsinki. 
 
This protocol and any amendments will be submitted to a properly constituted Independent Ethics 
Committee (IEC) or Institutional Review Board (IRB), in agreement with local legal prescriptions, for 
formal approval of the study conduct. The decision of the IEC/IRB concerning the conduct of the study will 
be made in writing to the investigator and a copy of this decision will be provided to the sponsor before 
commencement of this study. The investigator should provide a list of IEC/IRB members and their affiliate 
to the sponsor. 
 
It is expected that the IRB will have the proper representation and function in accordance with federally 
mandated regulations. The protocol, Investigator’s Brochure, informed consent, advertisements (if 
applicable), written information given to the patients, safety updates, annual progress reports , and any 
revisions to these documents will be provided to the IRB/IEC by the investigator. 
1.1 Subject Information and Consent 
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study. See 
Attachments 1, 2, and 3 for copies of the Subject Informed Consent Form. This consent form will be 
submitted with the protocol for review and approval by the IEC/IRB for the study. The formal consent of a 
subject, using the IEC/IRB-approved consent form, must be obtained before that subject undergoes any 
study procedure. The consent form must be signed by the subject or legally acceptable surrogate, and 
the investigator-designated research professional obtaining the consent.  
 
A written informed consent form, which contained all US federally required elements, all ICH-required 
elements, and Health Insurance Portability and Accountability Act (HIPAA) Authorization information in 
language that was understandable to the subject, will be given to each subject. The process of obtaining 
the informed consent will be in adherence with all federal regulations, ICH requirements, and local laws.  
 
In obtaining and documenting informed consent, the investigator should comply with the applicable 
regulatory requirement(s), and should adhere to Good Clinical Practice (GCP) and to ethical principles 
that have their origin in the Declaration of Helsinki. 
 
Before recruitment and enrollment onto this study, the patient will be given a full explanation of the study 
and will be given the opportunity to review the consent form. Each consent form must include all the 
relevant elements currently required by the FDA Regulations and local or state regulations. Once this 
essential information has been provided to the patient and the investigator is assured that the patient 
understands the implications of participating in the study, the patient will be asked to give consent to 
participate in the study by signing an IRB-approved consent form prior to study participation. 
 
Prior to a patient’s participation in the trial, the written informed consent form should be signed and 
personally dated by the patient or the patient’s legally acceptable representative, and by the person who 
conducted the informed consent discussion. 
2 Introduction 
This is a single-center study of pharmacogenomic-guided tacrolimus dosing and monitoring in kidney 
transplant recipients. 


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2.1 Background and Rationale 
Renal transplantation from either a live or deceased donor has become the preferred treatment for 
patients with end-stage renal disease because it prolongs survival and improves quality of life (Port 1994; 
Schnuelle et al. 1998; Tonelli et al. 2011). Tacrolimus is the standard treatment for immunosuppression 
with kidney transplant patients as tacrolimus is associated with better graft survival and lower incidence of 
nephrotoxicity compared to cyclosporine (Bowman and Brennan 2008; Webster et al. 2005a; Webster et 
al. 2005b). 
 
Tacrolimus is a good candidate for pharmacogenomic-guided dosing and monitoring as it displays 
variable pharmacokinetics and is also a narrow therapeutic index drug, whereby low drug exposure 
increases risk to kidney rejection and high doses correlate with nephrotoxicity (Birdwell et al. 2015; 
Hesselink et al. 2014; Provenzani et al. 2013). Reaching and maintaining therapeutic tacrolimus levels is 
also often problematic despite adjustment of the initial dose to body weight and frequent monitoring of 
tacrolimus levels. Furthermore, response to tacrolimus is dose-dependent with large intra- and inter-
patient variability that has been partly attributed to genetic differences in the CYP3A5 gene (Birdwell et al. 
2015; Hesselink et al. 2014; Provenzani et al. 2013; Staatz, Goodman, and Tett 2010a, b). 
 
The CYP3A5 gene encodes for the drug-metabolizing enzyme CYP3A5, which is primarily responsible for 
the hepatic and renal clearance of tacrolimus (Dai et al. 2006). Specifically, the single nucleotide 
polymorphism (SNP), rs776746, results in a functional variation that substitutes an adenine (A) for a 
guanine (G) at position 6986 within intron 3 of the CYP3A5. A change from A to G at this position creates 
a cryptic splice site in intron 3 that results in an alternatively spliced isoform that has an insertion in intron 
3. This alternative spliced mRNA alters the reading frame and results in a premature termination codon 
that leads to a nonfunctional protein (Birdwell et al. 2015; Lamba et al. 2012; Staatz, Goodman, and Tett 
2010a, b). The nonfunctional CYP3A5 enzyme corresponds to the CYP3A5*3 allele, while the functional 
allele is considered CYP3A5*1. Patients who have one (CYP3A5*1/*3) or both copies of the functional 
allele (CYP3A5*1/*1) are considered CYP3A5 expressers. Due to high CYP3A5 expression, patients with 
CYP3A5*1/*1 or CYP3A5*1/*3 genotype may have lower levels of tacrolimus than patients with 
CYP3A5*3/*3 genotype, predisposing them to reduced immunosuppressive effects that may lead to acute 
kidney rejection. On the other hand, patients who have CYP3A5*3/*3 genotype do not express CYP3A5 
(CYP3A5 nonexpressers), which can lead to increased risk for toxicities due to supratherapeutic 
tacrolimus levels (Birdwell et al. 2015; Lamba et al. 2012; Staatz, Goodman, and Tett 2010a, b; van 
Gelder, van Schaik, and Hesselink 2014). In line with these findings, the Clinical Pharmacogenetics 
Implementation Consortium (CPIC) has recently published guidelines for CYP3A5 genotyping and 
tacrolimus dosing (Birdwell et al. 2015). 
 
The CYP3A5 6986A>G (rs776746) variant is ideal for a pharmacogenomic study as it has a high 
estimated allele frequency across various populations, including Whites (82% to 95%), Japanese (85%), 
Southeast Asians excluding Japanese and Chinese (67%), Chinese (65%), Pacific Islanders (65%), 
Southwest American Indians (40%), and African Americans (33%) (Lamba et al. 2012). The CYP3A5 
6986A>G genotype has been consistently associated with tacrolimus clearance and trough levels 
(Macphee et al. 2005; Macphee et al. 2002; Quteineh et al. 2008; Renders et al. 2007; Thervet et al. 
2003; Zhang et al. 2005; Zheng et al. 2003). In a recently published review and meta-analysis, the 
functional CYP3A5*1 allele was associated with higher risk of acute rejection and chronic nephrotoxicity 
(Rojas et al. 2015). 
 
Whether pharmacogenomic-guided dosing leads to improved clinical outcomes is yet to be determined A 
prospective study conducted in France showed that genotype-guided dosing resulted in quicker time to 
reach therapeutic tacrolimus levels but did not demonstrate improved clinical outcomes compared to 
standard dosing (Thervet et al. 2010). Furthermore, a recent study in Netherlands did not find any 
difference in the incidence of acute rejection or in the proportion of patients with sub or supratherapeutic 
tacrolimus levels when comparing subjects who received genotype-guided vs. standard tacrolimus dose 
(Shuker et al. 2016). Of note, a difference in tacrolimus trough levels and dose requirement was observed 
between CYP3A5 expressers and nonexpressers throughout the 3-month period (Shuker et al. 2016). In 


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both these studies, the population was primarily low-risk (living donor recipients) and was mostly 
Caucasian (90%). 
 
To further investigate if these results are relevant in the US kidney transplant population, particularly in 
centers comprising of at least 40% African Americans who are likely to carry the CYP3A5*1 (expressers) 
allele and be under-dosed, we are proposing a prospective, single-center study in the US that is aimed at 
evaluating the relationship between genotype-guided tacrolimus dosing, drug concentrations, and clinical 
outcomes. Unlike the study population in both European studies, the transplant recipients here at the 
University of North Carolina in Chapel Hill (UNC-CH) comprise of at least 40% African Americans, with 
majority of the kidney transplant recipients (75%) receiving their kidneys from deceased donors and are 
therefore more likely to be at risk for poorer outcomes. Unlike the standard tacrolimus dosing and target 
tacrolimus levels used by Thervet and colleagues in France, the tacrolimus dosing algorithm and 
therapeutic target levels being used at UNC-CH is more representative of the regimen for tacrolimus in 
the US kidney transplant recipients. Additionally, unlike the immunosuppressant regimen used by Thervet 
et al and Shuker et al, the immunosuppressant regimen that are currently being used at UNC-CH are 
representative of the current therapeutic management of US kidney transplant recipients. The standard 
tacrolimus dosing algorithm currently being used at UNC-CH was developed in accordance to current 
clinical guidelines and will be considered in the study as standard therapy. To provide a comparable 
pharmacogenomic-guided algorithm, the genotype-guided dosing algorithm described in Section 6.2.1 is 
similar to the one that was used in France (Thervet et al. 2010) and Netherlands (Shuker et al. 2016). 
Hence, the results of this study will provide more relevant findings for the US kidney transplant population 
as well as provide a reasonable comparison from previous and recently published pharmacogenomic 
studies in Europe. 
 
In addition, the results of this study will allow us to explore the feasibility of using genotype information for 
clinical decision making as well as the economic impact of using a genotype-guided treatment algorithm 
at UNC-CH kidney transplant unit. 
2.2 Previous Clinical Experience with Genotype-Guided Tacrolimus Dosing 
There are two large prospective pharmacogenomic studies that have tested the efficacy and safety of two 
genotype-guided tacrolimus doses: 0.15 mg/kg/day for CYP3A5 expressers and 0.3 mg/kg/day for 
CYP3A5 nonexpressers (Shuker et al. 2016). In both studies, there was no difference in safety outcomes 
between subjects who received standard tacrolimus dosing (0.2 mg/kg/day) versus those who received 
genotype-guided dosing (0.15 or 0.3 mg/kg/day). There were no significant between-group differences in 
the overall incidence and severity of biopsy-proven acute rejection (BPAR), rate of acute and delayed 
graft rejection, and incidence of adverse events, including infection, tacrolimus-associated nephrotoxicity, 
and post-transplant diabetes mellitus.  
 
Additionally, the genotype-guided dosing recommendation used in previous studies (Shuker et al. 2016), 
which is similar to the one proposed in this study protocol, follows the recommendation outlined in the 
Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline 
(https://www.pharmgkb.org/guideline/PA166124619). CPIC’s genotype-guided dosing recommendation 
for tacrolimus was classified as “strong" based on a combination of preclinical functional and clinical data 
as well as on some existing disease-specific consensus guidelines. 
 
Taken together, in addition to fact that genotype-guided dosing will only be given ONCE at the initial 
dosing of tacrolimus (subsequent dosing will then be based on tacrolimus trough levels and desired 
therapeutic concentration in accordance to standard procedure), the investigators do not anticipate any 
additional risks for subjects who will receive genotype-guided therapy compared to the standard 
tacrolimus dose that would have been given as part of their immunosuppressant regimen in the event that 
subjects do not consent to be included in the study. 
3 Study Objectives 
The primary objective of this study is to investigate the direct correlation of CYP3A5 genotype with 
tacrolimus trough levels and clinical outcomes. To meet this objective, a comparison of the primary and 


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secondary endpoints will be conducted between kidney transplant recipients who underwent prospective 
pharmacogenomic-guided tacrolimus dosing strategy (pharmacogenomic group) and patients who 
received standard tacrolimus dosing regimen historically and prospectively (control group). Patient data 
from the control group will be obtained from 2010 until the end of study period. 
 
The secondary objective of this study is to assess the economic benefit of genotype-guided tacrolimus 
dosing as measured by direct healthcare cost. 
 
Additionally, we will store banked DNA for future unspecified research from individuals who agree to 
participate in this study that are undergoing kidney transplantation at UNC-CH. A pharmacogenomic 
database will be created and maintained in a secure database for possible use in future studies to 
retrospectively correlate pharmacogenomics (SNPs and patterns of polymorphisms) with therapeutic drug 
monitoring levels and clinical metrics (eg., Quality/ Safety Metrics – mortality, morbidity, and other quality 
markers (eg., length of hospital stay, readmission rate etc). We plan to use genomic samples to base 
clinical care on genetic variants with known functional impact (ie, CYP2D6, TMPT, G6PD etc.). We 
therefore propose to study other smaller effect loci identified by association studies using the data for 
hypothesis generating analysis to correlate these single gene polymorphisms and polymorphism patterns 
with clinical outcomes by creating and then using a pharmacogenomic database. 
3.1 Primary Endpoint 
The primary endpoint of this study is to evaluate the proportion of patients reaching target levels (8-10 
ng/ml) on Day 3 and Day 7 after kidney transplantation. 
3.2 Secondary Endpoints 
The secondary endpoints are described below. 
• 
The incidence of BPAR within the first 3 months, 3-6 months, 6-9 months, and 6-12 
months after transplantation 
• 
Time to achieve tacrolimus therapeutic range at 0 to 4 months (8-10 ng/mL) 
• 
Proportion of patients who had dose adjustments and/or drug alteration or addition 
due to insufficient immunosuppression 
• 
Incidence of acute and chronic renal impairment. Renal function will be assessed 
using estimated Glomerular Filtration Rate (eGFR). Creatinine clearance (CrCl) may 
also be calculated as a reference. Patients will be categorized as having either mild 
(eGFR of 60 mL/min/1.73m2 to 89 mL/min/1.73m2), moderate (eGFR of 30 
mL/min/1.73m2 to 59 mL/min/1.73m2), or severe renal impairment (eGFR <30 
mL/min/1.73m2). 
• 
Incidence of adverse outcomes (ie, graft rejection, graft loss, renal impairment, 
adverse drug events, tacrolimus toxicities, and death) 
3.3 Exploratory Endpoints 
The exploratory endpoints relate to the secondary objective of this study and are described below. 
• 
Direct and indirect cost related to treatment and management kidney transplant 
recipients 
4 Study Design 
4.1 General Design 
This is a single-center study of pharmacogenomic-guided tacrolimus dosing and drug monitoring in 
kidney transplant recipients (Figure 1). The pharmacogenomic group will partake in a 12-month study 
comprising of two periods, Genotype-Guided Initial Dosing Intervention and Follow-up.  
 


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Following screening, acquisition of consent, and genotyping, a comprehensive medical chart review will 
be conducted for the control group. Twelve-month post-transplant data will be collected in the control 
group with timepoints that match with the pharmacogenomics group. 
 
Figure 1: Study Design 
 
 
Figure 1. This figure provides the general study design for the study, which comprises of the Screening 
and two study periods (Intervention and Follow-up) for the pharmacogenomic group. After consent and 
genotyping have been conducted, a comprehensive medical chart review will be conducted for the control 
group. 
 


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4.2 Rationale 
Due to cost, time constraints, and limited patient population (there are approximately 80 kidney transplant 
recipients at UNC-CH), we are not able to conduct the ideal randomized, double-blind, two-group parallel 
study. Rather, we are proposing a study design that is practical, given the available resources and still 
investigates the safety and effectiveness of using genotype-guided tacrolimus dosing. 
 
In this study, a comparison of the primary and secondary endpoints will be conducted between kidney 
transplant recipients who underwent prospective pharmacogenomic-guided tacrolimus dosing strategy 
(pharmacogenomic group) and patients who received standard tacrolimus dosing regimen historically and 
prospectively (control group). Although genotyping the control group can be more expensive and time 
consuming, knowing the genotype information of the control group will allow us to do the following: 1) 
identify if the control and pharmacogenomic groups are comparable with regards to genotype; and 2) 
conduct a subgroup analysis based on treatment and genotype, comparing the outcomes and tacrolimus 
levels of patients who are CYP3A5*1/*1, CYP3A5*1/*3, or CYP3A5*3/*3 who received standard therapy 
versus genotype-guided dosing. 
 
4.3 Patient Selection 
The study schemas for the pharmacogenomic and control groups are displayed in Figure 2. 
 
Figure 2: Study Schema 
 
 
 
Figure 2. The overall study schematic for the pharmacogenomics group is shown on the left, and the 
study schematic for the control group is shown on the right. 
 
Briefly, patients currently on the waitlist to receive kidney transplantation at UNC-CH who are being seen 
by the multi-disciplinary kidney transplantation team will be screened for eligibility. At the pre-intervention 
assessment (Study Day 0), buccal swab samples for genotyping will be collected on all eligible patients 
who provided informed consent (performed in real time). All buccal swab samples collected will then be 


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de-identified and sent to Cancer Genetics Incorporated (CGI) for CYP3A5 genotyping. Results of the 
genotyping test will be incorporated into electronic medical record (EMR) in addition to the genotype-
guided dosing recommendation for each patient. The initial tacrolimus dose will be based on the 
pharmacogenomic-guided treatment algorithm described in Section 6.2.1.. Eligible patients who 
consented to receive genotype-guided tacrolimus dose will enter the pharmacogenomic group and will 
receive the initial tacrolimus dosing based on genotype results following kidney transplantation (Study 
Day 1). Subsequent tacrolimus dosing will then be adjusted according to trough concentrations (C0) and 
therapeutic target concentrations of 8-10 ng/mL (0 to 4 months), 6-8 ng/mL (5 to 12 months), and 5-7 
ng/mL (>12 months). Dose adjustments and therapeutic drug monitoring will be conducted according to 
standard UNC-CH procedure. Hence, the genotype-guided dosing recommendation for tacrolimus only 
refers to the initial tacrolimus dose. All patients in the pharmacogenomic group will be followed from 
Study Day 2 and up to 12 months from the initiation of the first tacrolimus dose to assess long-term 
outcome (Follow-up Period). Safety and efficacy measures will be evaluated throughout the study per 
UNC-CH kidney transplant procedure. 
 
As the arrival of the donor kidney may come quickly and unexpectedly, it is possible that enrolled subjects 
may receive kidney transplantation prior to the incorporation of the CYP3A5 genotype result into EMR. In 
this case, patients will receive tacrolimus based on current standard tacrolimus dosing (0.1 mg/kg/day 
given in two divided doses) and will be included in the control group (prospective controls). Throughout 
the study, safety and efficacy measures will be evaluated in the prospective controls per UNC-CH kidney 
transplant procedure. These patients will also be followed from Study Day 2 and up to 12 months from the 
initiation of the first tacrolimus dose to assess long-term outcome (Follow-up Period). 
 
Additionally, age-, race-, and disease-matched patients who had previously received kidney 
transplantation with standard tacrolimus dosing will also be asked to give consent for genotyping 
(historical controls). These patients will also be included in the control group and their safety and efficacy 
data will be collected retrospectively for up to 12 months from the initiation of first tacrolimus dose. Hence, 
the control group will consist of both historical controls and potentially few prospective controls, if the 
scenario described above occurred. 
 
4.4 Genotyping and Dosing Administration 
The CYP3A5 6986A>G (rs776746) variant is ideal for a pharmacogenomic study as it has a high 
estimated allele frequency across various populations, including Whites (82% to 95%), Japanese (85%), 
Southeast Asians excluding Japanese and Chinese (67%), Chinese (65%), Pacific Islanders (65%), 
Southwest American Indians (40%), and African Americans (33%) (Lamba, 2012). At the UNC-CH multi-
disciplinary kidney transplantation team, we see approximately 80 patients for kidney transplantation per 
year. Of those 80 patients, approximately 60% are Whites and 40% are African Americans. We therefore 
expect that the CYP3A5 6986A>G (rs776746) variant will be relatively frequent in our study population. In 
addition, to be able to effectively compare outcomes between expressers and nonexpressers as well as 
between genotyped and nongenotyped subjects, we will make every effort to enroll African Americans to 
the study so as to get equal numbers of Caucasians and African Americans in our study. 
 
At study enrollment, buccal swab samples will be obtained for genotyping of the CYP3A5 6986A>G 
(rs776746) variant. Based on the genotyping result, patients will be categorized into two categories: 
• 
CYP3A5 expressers (CYP3A5*1/*3 and CYP3A5*1/*1) 
• 
CYP3A5 nonexpressers (CYP3A5*3/*3) 
 
Patients will then receive the initial tacrolimus dose based on the genotype-guided dosing algorithm 
outlined in Section 6.2.1. Subsequent tacrolimus dosing will then be adjusted according to trough 
concentrations and therapeutic target concentrations. 


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5 Subject Selection and Withdrawal 
5.1 Inclusion Criteria 
All new kidney transplant recipients aged 18 to 65 years who are admitted at UNC-CH and provided 
informed consent will be included in this study. 
5.2 Exclusion Criteria 
Patients will be excluded from participating in the study to receive genotype-guided tacrolimus dosing if 
he/she meets any of the exclusion criteria described below. 
• 
Recipients who did not consent to participate in the study. 
• 
Highly sensitized patients (ie, pretransplant T or B cell flow crossmatch positive) 
• 
Recipients of ABO incompatible kidney transplant 
• 
Recipients with preformed donor-specific antibodies (DSA) 
• 
HLA identical kidney transplant 
• 
Recipients of non-kidney transplant 
• 
Recipients of repeat transplant if they are on immunosuppression at the time of 
transplant 
• 
Patients using medications that have known pharmacokinetic (PK) drug interaction 
with tacrolimus 
• 
Patients in whom tacrolimus therapy is contraindicated 
5.3 Patient Recruitment and Screening 
Patients receiving pretransplant care for kidney transplantation at UNC-CH will be screened for eligibility 
based on the inclusion/exclusion criteria described in Sections 5.1 and 5.2. Following acquisition of 
consent, buccal swab samples will be collected for genotyping. Eligible patients who consented will 
receive genotype-guided dose for the initial tacrolimus dosing following kidney transplantation. 
Subsequent tacrolimus dosing will then be adjusted according to trough concentrations and therapeutic 
target concentrations. 
 
For the historical controls, age- and disease-matched patients who had previously received kidney 
transplantation with standard tacrolimus dosing will also be asked to give consent for genotyping 
(historical controls) during one of their post-transplant visit. Eligible patients who consented will be 
included in the control group and their safety and efficacy data will be collected retrospectively for up to 
12 months from the initiation of first tacrolimus dose. 
 
In addition, to be able to effectively compare outcomes between expressers and nonexpressers as well 
as between pharmacogenomic and control groups, we will make every effort to enroll African Americans 
to the study, including showing genuine interest to subjects’ well-being during one-on-one interactions, 
referring to them by their last names until given permission to use their first names, and using noninvasive 
means to collect samples (Johnson et al. 2011; Spence and Oltmanns 2011), so as to get equal numbers 
of Caucasians and African Americans in our study. 
5.4 Removal of Patients from the Genotype-guided Tacrolimus Therapy 
 
Removal of patients from the genotype-guided tacrolimus therapy can occur for the following reasons: 
 
1) Extraordinary medical circumstances: If, at any time the constraints of this protocol 
are detrimental to the patient's health, then the patient may be removed from the 
genotype-guided tacrolimus therapy. In this event, the investigator will do the 
following: 
• 
Notify the UNC-CH Principal Investigator 
• 
Document the reason(s) for withdrawal on flow sheets or Case Report Form 
(CRF) 


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• 
Notify the IRB 
 
2) Voluntary withdrawal 
 
3) Excessive toxicity: Patients who experience any grade III or grade IV AE deemed by 
the Principal Investigator to be related to tacrolimus therapy 
 
4) Investigator’s judgment: When deemed in the best interest of the patient by their 
attending physician, patients should be removed from the study and included in the 
intent-to-treat (ITT) analysis. 
 
5) Premature closure of the study: This study may be prematurely terminated, if in the 
opinion of the investigator there is sufficient reasonable cause. Written notification 
documenting the reason for study termination will be provided by the investigator. 
 
6) Circumstances that may warrant termination include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to patients 
• 
Failure to enter patients at an acceptable rate 
• 
Insufficient adherence to protocol requirements 
• 
Insufficient complete and/or evaluable data 
6 Study Drug  
6.1 Description 
Please refer to the current FDA-approved package insert (Attachment 4) for tacrolimus for clinical 
background information and possible side effects. 
6.2 Treatment Regimen 
All patients who consented will undergo genotype testing prior to transplant. The genotype-guided dosing 
recommendation as shown in Figure 3 below only refers to the initial tacrolimus dose. Subsequent 
tacrolimus dosing will then be adjusted according to trough concentrations and desired therapeutic 
concentrations. Therapy for oral tacrolimus will be prescribed by the kidney transplant team as part of a 
standard immunosuppression agent given to kidney transplant recipients starting at 0.1 mg/kg/day 
(nonexpressers) or 0.2 mg/kg/day (expressers) given in 2 divided doses; maximum total daily dose of 
tacrolimus is 20 mg/day. 
 
As the drug and the doses being used are not investigational, tacrolimus will be stored and dispensed in 
accordance to current UNC-CH pharmacy procedures. The patients and investigators will not be blinded 
in the study, hence there will be no procedure related to blinding of study drug. 
6.2.1 
Pharmacogenomic-guided dosing algorithm for the initial tacrolimus dose 
The pharmacogenomic-guided algorithm is described in Figure 3 below. The genotype-guided dosing 
recommendation only refers to the initial tacrolimus dose. Subsequent tacrolimus dosing will then be 
adjusted according to trough concentrations and desired therapeutic concentrations. 
 
Figure 3: Pharmacogenomic-guided Dosing Schematic 


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Figure 3 illustrates the genotype-guided dosing for the CYP3A5 expressers and nonexpressers for the 
initial tacrolimus dose following kidney transplantation. 
6.2.2 
Therapeutic Target Concentrations 
The drug will be adjusted and monitored using current UNC-CH standard kidney transplantation treatment 
procedure to reach the following therapeutic target levels: 
• 
8-10 ng/mL during 0 to 4 months of treatment 
• 
6-8 ng/mL during 5 to 12 months of treatment 
• 
5-7 ng/mL at >12 months of treatment 
 
Therapeutic drug monitoring, which provides a measure of compliance, will also be conducted according 
to standard UNC-CH procedure. 
7 Study Procedures 
Efficacy and safety measures will be assessed throughout the study. All study procedures will be 
conducted in accordance to applicable regulatory and ethical requirements.  
 
The schedule of events prior to pharmacogenomic intervention and during the Intervention and Follow-up 
periods are briefly described below and is summarized in Table 1. 
7.1 Pre-intervention Assessments 
The following procedures will be performed at Study Day 0, prior to initiation of the genotype-guided initial 
tacrolimus dose 
• 
Screening for eligibility 
• 
Acquisition of informed consent 
• 
Buccal swab sample collection 
• 
Hematology, clinical chemistry, urinalysis, eGFR, medical history, and physical exam 
Genotyped patients 
undergo kidney 
transplantation
CYP3A5*1/*1 and 
CYP3A5*1/*3 
(Expressers)
Initiate tacrolimus 0.2 
mg/kg/day in 2 
divided doses
CYP3A5*3/*3 
(Nonexpressers)
Initiate tacrolimus 0.1 
mg/kg/day in 2 
divided doses


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• 
Genotyping 
7.2 Pharmacogenomic Intervention 
On Study Day 1, Eligible patients who consented will receive genotype-guided dosing only for the initial 
tacrolimus dosing (0.1 mg/kg/day for CYP3A5*3/*3 or 0.2 mg/kg/day for CYP3A5*1/*1 or CYP3A5*1/*3). 
In addition, the following safety and efficacy parameters will be collected: hematology, clinical chemistry, 
urinalysis, adverse event (AE) and renal function assessments, medical history, physical exam, 
tacrolimus levels, and immunosuppressive and tacrolimus therapy modifications. 
7.3 Post-intervention or Follow-up Assessments 
Following administration of the initial genotype-guided tacrolimus dose, protocol-driven therapy will cease, 
and standard of care for tacrolimus dose adjustment and monitoring will be initiated. Efficacy and safety 
data, including AE assessments, renal function, and tacrolimus levels, will still be collected at each 
subsequent standard follow-up interval post Study Day 1 and up to 12 months of the initial tacrolimus 
dose. 
 


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Table 1: Schedule of Events 
 
Procedure 
Study 
Day 0 
Study 
Day 1 
3-day 
Follow-
up 
7-day 
Follow-
up 
Week 2 
Follow-
up 
Week 3 
Follow-
up 
Week 4 
Follow-
up 
3-month 
Follow-
up 
6-month 
Follow-
up 
9-month 
Follow-
up 
12-
month 
Follow-
up 
Screening 
x 
 
 
 
 
 
 
 
 
 
 
Informed 
consent 
x 
 
 
 
 
 
 
 
 
 
 
Buccal swab 
sample 
x 
 
 
 
 
 
 
 
 
 
 
Medical history 
x 
 
 
 
 
 
 
 
 
 
 
Drug therapy 
assessment  
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Physical Exam 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Clinical 
chemistry 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
CBCs with 
differentials 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Urinalysis 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
CrCl 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
eGFR 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Genotyping 
x 
 
 
 
 
 
 
 
 
 
 
Initiation of 
genotype-
guided dose 
 
x 
 
 
 
 
 
 
 
 
 
Tacrolimus 
level 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
AE 
assessments1 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Efficacy 
assessments2 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Abbreviations: AE=adverse event; BPAR = biopsy proven acute rejection; CBC = complete blood count; CrCl = creatinine clearance; eGFR = 
estimated Glomerular Filtration Rate. 
1The AE assessment will be conducted using the AE form (see AE Attachment). AEs may be assessed during an in-person clinic visit or a 
scheduled phone call. 
2Episodes of rejections will be assessed as part of the efficacy assessments. Information from biopsies, if collected, will also be assessed.


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8 Statistical Plan 
8.1 General Considerations 
This is a prospective, single-center study aimed to assess the efficacy and tolerability of the initial genotype-
guided tacrolimus dosing following kidney transplantation. Proper use of the study design will be ensured by 
the study investigators informing the study statistician, Dr. Thomas Urban. Additionally, quarterly follow-up on 
patient accrual will be initiated by the study investigators as further assurance of compliance with the design 
of the study laid out in this protocol. 
 
The term “pharmacogenomic group” will refer to prospective patients who received genotype-guided dose for 
the initial tacrolimus dose. The term “control group” will refer to age- and disease-matched eligible patients 
who received the standard tacrolimus dose after kidney transplantation from 2010 to the end of study period. 
 
8.2 Sample Size Determination 
The primary endpoint of this study is to evaluate the proportion of patients reaching target levels (8-10 ng/ml) 
on day 3 and day 7 after kidney transplantation.  
 
We calculated that a total study population of 260 patients (pharmacogenomics group = 130; control group = 
130 patients) would be sufficient to provide a statistical power of 90% to detect a significant difference 
between the two groups, on the basis of a two-sided χ2 test, a significance level of 5%, a frequency of 
approximately 50% for the CYP3A5*3/*3 genotype, and a 40% and 60% frequency of the primary endpoint in 
the control and pharmacogenomic group (Thervet et al. 2010), respectively. Because of the nature of the 
study design we anticipate a small rate of sample dropout (<5%), which should not appreciably impact the 
power of the study. 
8.3 Statistical Methods 
All analyses will be performed on the basis of the ITT principle. Unless stated otherwise, the term “descriptive 
statistics” will refer to the number of subjects, mean, median, standard deviation, minimum, and maximum for 
continuous data and to frequencies and percentages for categorical data. Categorical variables will be 
summarized as counts and percentages. All statistical parameters (eg, mean, median, standard deviation) 
will be tabulated together with their corresponding confidence intervals (CIs) and/or standard errors.  
 
Continuous variables will be summarized as medians with first to third quartiles. Appropriate conventional 
statistical analyses will be used to assess the primary and secondary endpoints. Categorical data will be 
compared using χ2 or Fisher’s exact test, as appropriate; some comparisons will require correction for 
potential confounders such as race/ethnicity; these will be performed using the Cochrane-Mantel-Haenszel 
test. Continuous variables will be compared using the Wilcoxon test or the t-test. The incidence of an adverse 
outcome (ie, graft rejection, graft loss, renal impairment, tacrolimus toxicities, and death) will be estimated 
using Kaplan–Meier curves. Depending on the distribution of the data, time-to-event (ie, time to reach 
therapeutic target range) analysis will be performed using an appropriate parametric, nonparametric, or semi-
parametric survival analysis method. An ANOVA model may be utilized and tabulated along with the CIs. 
During the analysis, if a possible confounding variable was identified, the variable will be measured and 
entered into the analysis as a covariate. 
 
Confounding factors that may impact tacrolimus response include race, age, and disease characteristics. It is 
possible that there may be differences in baseline demographics and disease states between the 
pharmacogenomic and control group. To minimize the potential impact of confounding variables on treatment 
response, an overall matching strategy has been incorporated into the design of the study. Please see 
Section 8.4 for more information regarding the matching strategy.  
 
The exploratory endpoints will be analyzed using descriptive statistics. No formal statistical testing is planned 
at this time. 
 


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Statistical testing, whenever performed, will be 2-sided with a significance (alpha) level of 0.05. In addition, 
95% confidence intervals will also be determined. All statistical analyses will be conducted using SAS® or 
other appropriate statistical programs. 
8.4 Matching Strategy 
As there are confounding variables, including age, race and disease state that may impact the results of the 
study, our study design incorporates an overall matching strategy, so that we can identify a well-matched 
control group. First, to control for differences in care over time, patients in the pharmacogenomic group will be 
matched to controls enrolled from 2010 to present. This time period was selected as there had been no major 
changes in standard of care or treatment regimen since 2010. After eligibility is met, control patients will be 
selected to match the pharmacogenomic group using a computerized matching algorithm that has been 
optimized to match baseline demographic and disease characteristics that have been identified a priori as likely 
to influence the treatment response to tacrolimus. To balance the trade-off between minimizing bias and 
maximizing matched sample size, a systematic approach will be conducted to identify the number of matched 
control patients for each patient in the pharmacogenomic group. This approach will include the following steps: 
1) run the desired matching algorithm, starting with 1:1 (one control to one patient in the pharmacogenomic 
group) matching and iterating until the maximum desired number of potential controls per treated subject is 
reached; 2) for each iteration, test for covariate balance; and 3) generate numeric summaries and graphical 
plots of the balance statistics across all iterations in order to determine the optimal number (Linden and 
Samuels 2013). 
 
The selection of patients for the control group using a matching algorithm will be conducted by an independent 
statistician in a blinded and unbiased manner. The statistician will have no knowledge of survival outcome, 
other outcome data, and genotype.  
 
9 Safety and Adverse Events 
Safety will be assessed throughout the study by evaluating physical exam, clinical chemistry, urinalysis, 
hematology, renal function, and incidence of adverse events (ie, graft rejection, graft loss, renal impairment, 
drug-related adverse events, tacrolimus toxicities, and death). 
9.1 Definitions 
Unanticipated Problems Involving Risk to Subjects or Others is defined as any incident, experience, or 
outcome that meets all of the following criteria:  
• Unexpected in nature, severity, or frequency (ie, not described in study-related documents 
such as the IRB-approved protocol or consent form, the investigators brochure, etc.) 
• Related or possibly related to participation in the research (ie, possibly related means 
there is a reasonable possibility that the incident experience, or outcome may have been 
caused by the procedures involved in the research, 
• Serious (as defined below); “serious” is different than “severe” as reported in the NCI 
CTCAE criteria, which applies a grade to the AE. 
 
Adverse Event 
An AE is any symptom, sign, illness or experience that develops or worsens in severity during the course of 
the study. Intercurrent illnesses or injuries should be regarded as AEs. Abnormal results of diagnostic 
procedures are considered to be AEs if the abnormality: 
• results in study withdrawal 
• is associated with an SAE 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
 
Serious Adverse Event 
All AEs are classified as serious or nonserious. A serious AE (SAE) is any AE that is:  
• fatal 
• life-threatening 
• requires or prolongs hospital stay 


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• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
 
Important medical events are those that may not be immediately life threatening, but are clearly of major 
clinical significance. They may jeopardize the subject, and may require intervention to prevent one of the 
other serious outcomes noted above. For example, drug overdose or abuse, a seizure that did not result in in-
patient hospitalization, or intensive treatment of bronchospasm in an emergency department would typically 
be considered serious.  
 
All AEs that do not meet any of the criteria for serious are regarded as nonserious AEs.  
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study. A preexisting condition should be 
recorded as an AE if the frequency, intensity, or the character of the condition worsens during the study 
period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting condition. At the end 
of the study, any new clinically significant findings/abnormalities that meet the definition of an AE must also 
be recorded and documented as an AE.  
 
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an AE if any one of the following conditions is met:  
• The laboratory abnormality is not otherwise refuted by a repeat test to confirm the 
abnormality 
• The abnormality suggests a disease and/or organ toxicity 
• The abnormality is of a degree that requires active management; e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic 
investigation, etc. 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any AE that results in hospitalization or prolonged hospitalization should be documented and reported as an 
SAE unless specifically instructed otherwise in this protocol. Any condition responsible for surgery should be 
documented as an AE if the condition meets the criteria for an AE.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an AE in the 
following circumstances: 
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for a preexisting condition. Surgery should not be reported as an outcome of an AE if the 
purpose of the surgery was elective or diagnostic and the outcome was uneventful. 
• Hospitalization or prolonged hospitalization required to allow efficacy measurement for the 
study. 
• Hospitalization or prolonged hospitalization for therapy of the target disease of the study, 
unless it is a worsening or increase in frequency of hospital admissions as judged by the 
clinical investigator. 
9.2 Recording of Adverse Events 
AEs will be collected following kidney transplantation. AEs are recorded as part of routine standard of care for 
kidney transplant recipients. 
 
At each contact with the subject, the investigator must seek information on AEs by specific questioning and, 
as appropriate, by examination. Information on all AEs should be recorded immediately in the source 
document. All clearly related signs, symptoms, and abnormal diagnostic procedures results should recorded. 
 
All AEs occurring during the study period must be recorded. The clinical course of each event should be 
followed until resolution, stabilization, or until it has been determined that the study treatment or participation 
is not the cause. SAEs that are still ongoing at the end of the study period must be followed up to determine 


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the final outcome. Any SAE that occurs after the study period and is considered to be possibly related to the 
study treatment or study participation should be recorded and reported immediately. 
 
All AEs, both serious and nonserious, and deaths that occur during the patient’s study participation will be 
recorded. All SAEs should be monitored until they are resolved or are clearly determined to be due to a 
patient’s stable or chronic condition or intercurrent illness(es). 
9.3 Reporting of Serious Adverse Events and Unanticipated Problems 
Investigators must conform to the AE reporting timelines, formats, and requirements of the various entities to 
which they are responsible, but at a minimum those events that must be reported are those that are: 
• related to study participation, 
• unexpected, and  
• serious or involve risks to subjects or others (see definitions, Section 9.1).  
9.3.1 
Investigator reporting 
For reportable deaths, the initial submission to the IRB may be made by contacting the IRB Director or 
Associate Director. The AE/Unanticipated Problem Form is required as a follow up to the initial submission. 
 
All AEs may be submitted on FDA Form 3500A or in a narrative format. If supplied as in a narrative format, 
the minimum information to be supplied is noted above at the beginning of Section 9.3.  
 
Any serious or unexpected event, which occurs to any patient in the course of their treatment on this study or 
within 30 days following cessation of treatment, must be reported immediately within 24 hours of the 
investigator learning of its occurrence. The immediate reports should be followed promptly by detailed, written 
reports. The Principal Investigator is responsible for reporting the SAEs to the FDA in accordance with 21 
CFR 312.32 and 21 CFR 314.80. If required, an NCI Adverse Drug Reaction (ADR) form will be completed 
and copies sent to: 
• 
Principal Investigator 
• 
IRB 
• 
UNC-CH Investigational Drug Service 
• 
Protocol Office ADR file 
 
For both serious and nonserious AEs, the investigator must determine both the intensity of the event and the 
relationship of the event to drug administration. 
 
• 
Relationship to drug administration will be determined by the investigator responding yes 
or no to the question: Is there a reasonable possibility that the AE is associated with the 
drug? 
• 
Intensity for each AE, including any lab abnormality, will be determined by using the NCI 
CTCAE, version 3.0, as a guideline, wherever possible. 
(http://ctep.cancer.gov/forms/CTCAEv3.pdf) 
9.4 Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study. This safety monitoring 
will include careful assessment and appropriate reporting of AEs as noted above, as well as the construction 
and implementation of a study management plan. Please see Section 11 for more information. Medical 
monitoring will include a regular assessment of the number and type of SAEs. 
9.5 Interim Analysis 
An interim analysis will be performed on the primary endpoint when 30% of eligible patients have been enrolled 
and have completed the 6 months follow-up. The interim analysis will be performed by an independent 
statistician who is blinded for the subjects’ treatment allocation. 
 
Some of the questions that will be evaluated during the interim analysis will include: 
 
• 
Is there an unexpectedly high rate of severe or life-threatening adverse events? 
• 
Is the outcome of the trial treatment comparable with that of previous studies? 


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• 
Do the results of the interim analysis prove statistically significant differences between the trial 
treatments that exceed the differences defined by the statistical guidelines of the trial? 
10 Data Handling and Record Keeping 
10.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to HIPAA requirements of 
1996. Those regulations require a signed subject authorization informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, retains 
the ability to use all information collected prior to the revocation of subject authorization. For subjects that 
have revoked authorization to collect or use PHI, attempts should be made to obtain permission to collect at 
least vital status (ie, that the subject is alive) at the end of their scheduled study period. 
 
Of major concern to most research participants is the confidentiality of information collected as part of a 
research study. All information will be kept strictly confidential and used for research purposes only. No 
identifying information will be disclosed in reports, publications, or presentations. 
 
Procedures to assure confidentiality will be strictly observed. The study will follow standard guidelines to 
assure that participant confidentiality is maintained. All data will be:  
• 
Kept in confidential locked files; 
• 
Identified by participant identification number (PID) only, which will be generated using 
random alpha numeric code only; and 
• 
Kept separately from identifying information used for subject tracking and follow-up 
contacts. 
 
The following methods will be utilized to protect the confidentiality of participant data:  
1) Data collected at clinical site and entered into the database will contain a PID number 
that does not reflect any personal information.  
 
2) Data linking a participant to a PID number will be stored locally in locked files. This 
information will not leave the clinical site and will be accessible to clinical site study staff 
only (but IRB may request access to patients’ files during site visits). 
 
3) Access to the data will be strictly controlled. User names and passwords will be 
distributed only to the appropriately trained clinical staff members. 
 
4) Specimens collected from participants and transferred to the CGI will be coded for 
tracking purposes but will not contain any personal identifiers.  
 
5) The statistician or the primary person responsible for the data analysis will not receive 
any original data for auditing or quality assurance purposes that contain any personal 
identifying information. Any personal identifiers will be completely removed prior to 
sending the data. 
10.2 Records Retention 
Methods for record retention are stated in the clinic’s SOPs. 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 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. 
 


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The patient’s confidentiality will be maintained and will not be made publicly available to the extent permitted 
by the applicable laws and regulations. 
10.3 Method of Data Collection 
Initially, while kidney transplant recipients receive care in the hospital, all data will be collected from medical 
and drug records obtained during the inpatient hospitalization. Subsequently, data on clinical, medication use, 
and environmental factors will be collected post-transplant in the clinic by the renal transplant coordinator who 
enters the patients’ clinical findings, including phone conversations, in EMR.  
 
10.4 Data Obtained for Analysis 
10.4.1 Data to be collected 
There will be 3 broad categories of data collected 1) patient-specific clinical and environmental data 2) 
genotype data, and 3) study outcomes. All participants will undergo a baseline interview to collect clinical and 
environmental data, and buccal swab sample will be collected for DNA extraction, genotyping, and storage 
for pharmacogenomic studies. At each subsequent visit, participants will undergo a follow-up interview as 
part of routine care to collect clinical, environmental, and outcome data. 
10.4.1.1 Clinical and Environmental Factors  
The clinical and environmental factors to be collected will be those that can alter the response to tacrolimus, 
increase the risk of any of the other study outcomes (eg, graft rejection), and fully describe the patient 
population and relevant subgroups. 
 
Clinical factors include factors associated with altered tacrolimus dose such as age, sex, pre-existing medical 
conditions, and body surface area. Environmental factors include interacting medications, smoking, and 
alcohol use. Interacting medications will include all possible interacting medications and dietary supplements 
that may alter the PK or pharmacodynamics (PD) of tacrolimus. All information pertaining to clinical and 
environmental factors can be found in the EMR as it is routinely collected as standard care for kidney 
transplant recipients. 
10.4.1.2 Genetic Data 
There will be two types of genetic data: 1) genotype data related to tacrolimus dosing, which includes primary 
genotype data required for the dosing algorithms and 2) other genetic data that will be used for additional, 
ancillary analyses. The data unrelated to the dosing algorithms will be derived from the stored buccal swab 
sample at the Central Laboratory (CL) and are not required for the clinical trial itself. As such, they are not 
discussed further in this protocol. The first (genotype data required for the dosing algorithm) will be assayed 
at CGI under the strictest CLIA (Clinical Laboratory Improvement Amendment) standard for genotyping of 
human specimens. The other genotype/sequencing data will be used to define additional genetic 
determinants of tacrolimus dosing in patients who have disparate response from the genotype-guided dose.  
 
Genotyping at the Central Laboratory. The CL will be responsible for: 1) extracting DNA from all participants’ 
buccal swab samples and 2) storing DNA on all participants for future genotyping of other variants of other 
genes that may be important for assessing response to tacrolimus. For future studies, the CL will be 
responsible for developing a multiplex platform that can assay numerous genetic variants within one reaction. 
 
Sample Collection for Storage and Analyses in Central Laboratory. Following acquisition of informed consent, 
buccal swab samples will be collected per manufacturer’s directions. Using DNA isolation kits suitable for 
buccal swab samples, DNA will be isolated at the CL according to the manufacturer’s instructions. A third of 
the aliquot will be retained by the CL, and the remainder will be delivered to CGI in a chilled insulated 
container. The DNA samples will be stored in barcoded tubes at the CL. All DNA samples that will be sent to 
CGI will also be in barcoded tubes, so that laboratory personnel at CGI are blinded to the patient’s identity. All 
samples will be tracked using the barcode and tracking system. 
10.4.2 Historical Data 
Historical data from the control group will be obtained from 2010 until the end of study period. The historical 
controls will consist of age- and disease-matched patients who met eligibility criteria and have had previously 


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received kidney transplantation with standard tacrolimus dosing. These patients will also be asked to give 
consent for genotyping during a standard routine follow-up visit post-transplantation. Eligible patients who 
consented will be included in the control group and their safety and efficacy data will be collected 
retrospectively for up to 12 months from the initiation of the first tacrolimus dose. 
10.4.3 Missing Data 
The main problem from missing data will be missing tacrolimus blood levels either from attrition or missed 
visits. Given that the initial monitoring of tacrolimus (Day 3 and Day 7) for the primary outcome will be 
conducted at the hospital, we expect the issue of attrition and missing data to be very unlikely. Extensive 
efforts will be made to collect complete information on each subject enrolled throughout the study. The 
optimal approach to missing data is to be proactive and be diligent in avoiding it. Additional methods, 
including calendar alerts, e-mail reminders, and reminder calls may be employed to minimize missed visits. 
10.5 Data Management 
Using appropriate data management tools such as Research Electronic Data Capture (REDCap), the data 
management team will develop a data validation plan, rule set specifications, and programming logic to 
implement data validation rules. The rule set will include checks for missing fields, range checks, skip pattern-
logic, and inter and intra form checks. Prior to analysis of the data, the data management team will perform 
extensive, independent testing of the validation program functionality by entering data known to violate 
validation rules and determining if the errors are detected. 
 
Data validity will be monitored to address critical missing, inaccurate, illogical, or inconsistent values in the 
data submitted by the investigator and CL. Rules will be developed and applied to evaluate data in a 
hierarchical fashion: 1) safety and efficacy; 2) eligibility; 3) analysis and outcome data; and 5) descriptive 
non-outcome data. Rules will be implemented after the data are committed and are managed via the query 
tracking system. 
11 Study Management 
11.1 Study Monitoring Plan 
This study will be monitored by the investigator. The investigator will permit study-related monitoring, audits, 
and inspections by the IRB, government regulatory bodies, and University compliance and quality assurance 
groups of all study related documents (eg, source documents, regulatory documents, data collection 
instruments, study data etc.). The investigator will ensure the capability for inspections of applicable study-
related facilities (eg, pharmacy, diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable University compliance and quality assurance offices. 
11.2 Central Laboratory Compliance 
 
The CL and CGI adhere to Good Laboratory Practice (GLP) guidelines for internal consistency and accuracy 
of data for analyses. As the services provided by CGI and CL are highly specialized, an experienced lab 
investigator or external subject matter expert will be identified to accompany any laboratory visit. The 
following activities may occur: 1) inspection of laboratory facilities, including adequacy and security of storage 
space, monitoring equipment for freezer function, certification documents, hardware and software for 
inventory control; 2) review of current GLP, laboratory Standard Operating Procedures (SOPs), quality 
assurance and specimen tracking procedures; 3) review process for receiving and processing a typical 
specimen; 4) review quality assurance documentation and procedures that assures adherence to protocol by 
laboratory personnel; 5) assess methods for communication of lab specimen preparation and transfer; 6) 
evaluate lab procedures for maintaining records, data storage, data transmission and security; 7) examine 
methodology for transmission of lab data and quality control procedures regarding the data transfer process; 
and 8) review methods for training and documenting competency of core lab staff.  
11.3 Patient Adherence with Study Medication 
Compliance to tacrolimus will be assessed through measurements of tacrolimus blood levels, which are 
routinely done as standard of care.  


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11.4 Deviations from the Protocol 
The investigator may implement a deviation from, or a change of, the protocol to eliminate an immediate 
hazard(s) to trial subjects without prior IRB/IEC approval/favorable opinion. As soon as possible, the 
implemented deviation or change, the reasons for it, and, if appropriate, the proposed protocol amendment(s) 
should be submitted to: 
• 
IRB for review 
• 
to the regulatory authority(ies), if required  
• 
to the sponsor for agreement (if applicable) 
11.5 Adherence to the Protocol 
Except for an emergency situation in which proper care for the protection, safety, and well-being of the study 
patient requires alternative tacrolimus dose, the study shall be conducted exactly as described in the 
approved protocol. Any deviation from the protocol must have prior approval by the Principal Investigator and 
must be recorded and explained. 
11.6 Amendments to the Protocol 
Should amendments to the protocol be required, the amendments will be originated and 
documented by the Principal Investigator at UNC-CH. The written amendment will be sent to 
investigators and must be submitted to the IRB. It should also be noted that when an amendment to the 
protocol substantially alters the study design or the potential risk to the patient, a revised consent 
form might be required. 
11.7 Obligations of Investigators 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in accordance with Title 
21 of the Code of Federal Regulations and/or the Declaration of Helsinki. The Principal Investigator is 
responsible for personally overseeing the treatment of all study patients. The Principal Investigator must 
assure that all study site personnel, including sub-investigators and other study staff members, adhere to the 
study protocol and all FDA/GCP regulations and guidelines regarding clinical trials both during and after study 
completion. 
12 Study Finances 
12.1 Funding Source 
This study will be funded internally through the Center for Pharmacogenomics and Individualized Therapy 
(CPIT). 
12.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or financial gain 
greater than the minimum allowable by their institution, etc.) will have the conflict reviewed by a properly 
constituted Conflict of Interest Committee with a Committee-sanctioned conflict management plan that has 
been reviewed and approved by the study sponsor prior to participation in this study. All UNC-CH 
investigators will follow the University conflict of interest policy. 
12.3 Subject Stipends or Payments 
No compensation for participation will be provided. The genotyping cost will be covered by funds available 
through CPIT. If the patient’s insurance does not cover the treatment dose (most will as it is an FDA 
approved dose), then we have funds available to make up the cost difference. 
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ATTACHMENT 1:  Adverse Event Assessment Form: