Trial Information

Tyrosine Kinase Inhibitors of BCR/ABL: Pharmacokinetic and Pharmacogenetic Study in Patients Affected by Chronic Myeloid Leukemia. Evaluation of Efficacy and Tolerability

1. Patients

1.1. Patients

CML patients will be included according to the following inclusion criteria: a) patients of
both sexes, b) age between 18 and 80 years, c) treated with imatinib or nilotinib, d)
included in the follow-up in the Divisions / Units of Hematology involved in the project e)
able to give informed consent to trial participation, f) with a proved compliance with the
scheduled treatment.

Patients will be excluded from participation to the study if: a) age <18 or >80 years or b)
unable to provide informed consent. The inability to attend the follow-up visits will not be
considered an exclusion criterion from the study. In this case, collected data will be used
in pharmacokinetic and statistical analyses on the basis of an intention-to-treat criterion.

The following conditions should be noted:

1. the concomitant administration of other drugs will be allowed, provided it will be
known the active ingredient, the dose and the period of administration;

2. smoking and herbal products will be allowed but they should be reported on patient's
case report form;

3. alterations in liver and kidney functions, body mass index higher than 28, or any other
alteration in physicochemical exams do not represent exclusion criteria.

1.2. Sample size

For the purposes of this study it has been estimated that it will need to recruit at least
206 subjects per drug (total, 412 patients), taking into account:

1. the minor allele frequency of the most studied polymorphism evaluated in this research
protocol (i.e., 0.3, T allele of the ABCB1 c.3435C>T polymorphism);

2. the 1:1 ratio allele C carriers and patients homozygous for the T allele of the ABCB1
c.3435C>T polymorphism;

3. covariate effects on drug pharmacokinetics will be considered significant when the mean
difference in pharmacokinetic parameters will be at least 20%;

4. the power of the study will be 80%;

5. the alpha error will be set at 0.05.

1.3. Enrollment

Patient enrollment in the various participating centers will be competitive, up to the
achievement of 206 individuals per drug.

At the first visit, the TIKlet study will be presented to patients who will satisfy the
inclusion / exclusion criteria. The following activities will take place:

- patients will be informed about the characteristics, objectives, and procedures of
the study.

- Consent to study participation will be confirmed by signing the informed consent
form.

- Personal alphanumeric code assignment for patient's identification and privacy.

- Updating of the enrollment list.

- Collection of main data of the patient (age, sex, age at diagnosis, clinical
chemistry test results as reported in the patient's record and any combination
therapies, with their doses and duration) within the individual case report form
(CRF).

- A blood withdrawal (approximately 4 mL) will be performed from a peripheral vein
of the forearm. The time at which blood will be withdrawn will be registered
within patient's CRF, together with the time elapsed from the last administration
of imatinib or nilotinib. For sample handling see section 2.1. "Blood sample
handling".

- The updated list of enrolled patients and individual case report forms will be
sent to the Division of Hematology, Dept. of Clinical and Experimental Medicine,
University of Pisa

1.4 Follow-up visits

According to clinical routine at each participating center, the following activities
should be performed during each follow-up visit:

- Collection of patient's data: clinical chemistry test results, cytogenetic
analysis and molecular biology results (BCR/Abl transcript levels), any
concomitant therapy, any adverse drug reaction.

- A blood sample (approximately 4 mL) will be collected from a peripheral vein of
the forearm. The time at which blood will be withdrawn will be registered within
patient's CRF, together with the time elapsed from the intake of last imatinib or
nilotinib dose. For sample handling see section 2.1. "Blood sample handling".

2. Laboratory analyses

Laboratory analyses will be conducted at the Division of Pharmacology, Department of
Clinical and Experimental Medicine, University of Pisa (measurement of drug plasma
concentrations, pharmacogenetic analyses) and at the Department of Pharmacology,
University of Bologna (pharmacogenetic analyses).

2.1. Blood sample handling

Each tube containing the patient's whole blood, plasma or DNA (see below) and used for
the execution of therapeutic monitoring or pharmacogenetic analyses will be identified
by a) the patient's code and b) the date of execution of blood withdrawal.

All blood samples will be collected in lithium-heparin Vacutainer® tubes, then they
will be stored at 4 °C until centrifugation. The sample will be centrifuged and the
resulting plasma (1.5 mL) will be collected and stored in a tube for therapeutic drug
monitoring. Only during the enrollment visit, an aliquot of whole blood (0.5 mL) will
be collected in a sterile eppendorf for molecular analyses. Both samples will be stored
at -20 °C for a maximum of 2 weeks, then they will be sent to the Dept. of Clinical and
Experimental Medicine, University of Pisa.

For sample storage see section 2.7. "Biological sample storage".

2.2 Measurement of plasma concentration of drugs

The measurement of plasma concentrations of imatinib and its active metabolite, and
nilotinib will be conducted by a high-performance liquid chromatography (HPLC) method
using commercially available kits (Chromsystems GmbH, Munich, Germany) on Waters Breeze
and Alliance instruments (Waters, USA).

Briefly, solid-phase extraction columns will be balanced with Equilibration buffers 1
and 2, then the internal standard and plasma (0.5 mL) will be added. The column will be
centrifuged, and subsequently washed with Wash buffer and water for HPLC. The eluate
will be collected by centrifugation, diluted with water for HPLC and injected (0.025
mL) into the HPLC system for the execution of the analysis. The peak area of the
analytes of interest (imatinib and its active metabolite, nilotinib and internal
standard) will be recorded and the actual concentration of drug in plasma will be
obtained by comparison with standard calibration curves for each day of analysis.

The HPLC analyses will be carried out on a weekly basis. The reports of the analysis
will be sent to the Divisions / Units of Hematology and registered into both the
patient's medical record and study CRFs.

2.3. Pharmacokinetic analyses

Plasma concentrations of imatinib/active metabolite and nilotinib will be analyzed
trough a population pharmacokinetic analysis according to a nonlinear mixed-effects
modeling approach, using the NONMEM software, version 7.2. The Xpose and PsN packages
will be used to check database, for model diagnostic and covariate testing. Former
models for imatinib/metabolite and nilotinib will be 1- and 2-compartment models with
first-order elimination and parameterized in terms of apparent drug clearance (CL/F),
volume of distribution (V/F) and absorption constant (ka), while the residual error
will be modeled as additive, proportional or mixed (additive and proportional) error.
The availability of records, goodness-of-fit plots and the precision of parameter
estimates, together with a generalized additive modeling procedure (GAM) implemented in
the Xpose package, will be adopted for model development and to explore correlations
among the covariates. In particular, age, weight, body mass index, liver (ALT, AST) and
renal function (creatinine plasma concentration), albumin and α1-acid glycoprotein
plasma concentrations and results of pharmacogenetic analyses will be considered as
possible covariates. A stepwise covariate model building will be performed with forward
inclusion and backward exclusion. In particular, a decrease in the objective function
value (OFV) greater than 3.84 points (i.e., p<0.05) and 6.63 points (i.e., p<0.01) will
be the criteria used in the forward inclusion and backward exclusion steps,
respectively. The goodness of the final model will be checked by bootstrap analysis
using the PsN and Xpose packages and calculating η-shrinkage. The area under the plasma
concentration-time curve from time zero up to infinity (AUC) and terminal elimination
half-life (t1/2) will be calculated from the individual empirical Bayes estimates (EBE)
from the final population pharmacokinetic model as follows:

AUC=(Total daily dose)/(CL/F) t1/2=ln(2)/kel where kel is imatinib elimination
constant.

2.4. Pharmacogenetic analyses

The aliquot of frozen whole blood will be employed for the extraction of genomic DNA
with a suitable kit (Qiagen, Milan, Italy) following manufacturer's instructions and
the nucleic acid will be stored at -80 ° C until the time of analysis in sterile tube
bearing the identification code of the patient.

Single nucleotide polymorphisms (SNPs) of the following genes will be assayed: ABCB1,
ABCG2, hOCT1, OCTN1, OATP1A2. SNPs will be evaluated by specific kits from Applied
Biosystems (Life Sciences, Milan, Italy) on an ABI Prism 7900HT Sequence Detection
System (Life Sciences). Each analysis will be performed in triplicate. Genotypes will
be automatically identified through the software SDS (Life Sciences). Frequency of
alleles and genotypes according to the Hardy-Weinberg law, haplotype and linkage
disequilibrium analysis will be performed using Arlequin software package.

2.5. Treatment effectiveness and tolerability

The response to the therapy with imatinib will be evaluated in agreement with criteria
recently published and adopted. In particular, results from chemical, biochemical and
molecular analyses will be performed every 2 weeks during the first 3 months of
treatment, then cytogenetic (metaphase chromosomal banding assay) and molecular
analyses (quantification of BCR-ABL transcription levels in plasma by real-time-PCR)
were performed every 6 and 3 months, respectively, until the attainment of a complete
response. The time to complete cytogenetic response (CCyR) and to major molecular
response (MMR) will be defined as the length of time elapsed from diagnosis and
response attainment for every patient.

Furthermore, treatment-associated adverse reactions (i.e., nausea and vomiting,
periorbital oedema, cramps and myalgia, neutropenia, skin and liver toxicities) will be
identified and scored according to the Common Toxicity Criteria-NCI grading system
(version 3).

2.6. Statistical analysis

The results will be analyzed in aggregate form. Parameter of dispersion (standard
deviation, range), central indexes (mean, median) and distribution (i.e.,
Kolmogorov-Smirnov test) will be calculated. The most appropriate statistical tests
(ANOVA, Fisher test, etc.) will be applied to investigate any possible difference
between groups, and the level of significance will be set at p = 0.05. The
identification of cut-off values for parameters predictive of efficacy / tolerability
(plasma concentrations, polymorphisms) will be pursued trough ad hoc analyses (receiver
operating characteristics analysis, evaluation of positive and negative predictive
values).

2.7. Biological sample storage

All biological samples [whole blood (0.5 mL), plasma (1.5 mL) and genomic DNA] will be
stored at the Division of Pharmacology, Department of Clinical and Experimental
Medicine, University of Pisa. Aliquots of DNA samples for pharmacogenetic analyses will
be stored at the Department of Pharmacology, University of Bologna.

Each sample will be identified by the individual alphanumeric code assigned to every
patient. According to protocol, patients may require the destruction of their own
biological samples.

2.8. Authorship

The criteria declared by the ICMJE (International Committee of Medical Journal Editors)
will be adopted for authorship definition.