determination of deferasirox plasma concentrations: do gender, physical and genetic differences...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ejh.12419

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Article Type: Original Article

Accepted date: 24-Jul-2014

Title: Determination of deferasirox plasma concentrations: do gender, physical and genetic

differences affect chelation efficacy?

Authors: F Mattiolia, M Puntonib, V Marinia, C Fucilea, G Milanoa, L Robbianoa, S Perrottac, V. Pintod, A

Martellia, G L Fornid

Affiliation:

aDepartment of Internal Medicine, Clinical Pharmacology and Toxicology Unit, University of Genoa,

Genoa, Italy

bClinical Trial Unit, Scientific directorate, E.O. Galliera, Genoa, Italy

cDepartment of Pediatrics, Second University of Naples, Naples, Italy

dSSD Ematologia – Centro della Microcitemia E.O. Galliera, Genoa, Italy

Running Title: Gender and physical differences affect deferasirox

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Corresponding author: Prof. Francesca Mattioli

aDepartment of Internal Medicine, Clinical Pharmacology and Toxicology Unit, University of Genoa,

Genoa, Italy

Address: Viale Benedetto XV, n. 2. I-16132 Genoa, Italy

Phone: +390103538850; Fax: +390103538232

Email: [email protected]

Abstract

Objectives. Bioavailability of deferasirox (DFX) is significantly affected by timing of administration

relative to times and to composition of meals. Its elimination half-life is also highly variable – in some

patients as a result of gene polymorphisms. Understanding if deferasirox plasma levels are related to

specific characteristics of patients could help physicians to devise a drug regimen tailored the

individual patient.

Methods. We analysed deferasirox plasma concentrations (CDFX) in 80 patients with transfusion-

dependent anemias, such as thalassemia, by a HPLC assay. We used a multivariate linear regression

model to find significant associations between CDFX and main patients clinical/demographical

characteristics of patients. All patients were genotyped for UGT1A1.

Results. Fifty-six patients were female and 24 were male, the great majority (88%) affected by β-

thalassemia, 15 were children and adolescents. No statistical correlation was detectable between

CDFX and DFX dose (p=0.6). Age, time from last drug intake to blood sampling and ferritin levels in the

6 months before study initiation were significantly and inversely associated with CDFX in univariate

analysis. In the multivariate analysis the only two factors independently and inversely associated with

CDFX levels were time from last drug intake to blood sampling and ferritin levels (p=0.006). A

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significant inverse correlation (p=0.03) was observed between CDFX and UGT1A1*28 gene

polymorphism, but only in patients with levels of lean body mass (LBM) below the median (p for

interaction=0.05).

Conclusions. The results could indicate that a higher plasma DFX concentration could be

associated with greater chelation efficacy. Since a correlation between dose and CDFX was not

demonstrated, it seems useful to monitor the concentrations to optimize and determine the

most appropriate dose for each patient. Interesting results emerged from the analysis of

genetic and physicals characteristics of patients: LBM was a borderline significant effect

modifier of the relationship between UGT1A1 polymorphisms and CDFX. Individual patient-

tailored dosing of DFX should help to improve iron-chelation efficacy and to reduce dose-

dependent drug toxicity.

Key words. Deferasirox, bioavailability, thalassemia, iron chelation therapy, therapeutic drug

monitoring.

Abbreviations: BMI, Body mass index; DFX, Deferasirox; HPLC, high performance liquid

chromatography; LBM, lean body mass.

Introduction

The therapeutic benefit of iron chelation (i.e. forced elimination of iron) with deferoxamine (DFO)

has been clearly established in more than 40 years of clinical practice. For patients with thalassemia,

its introduction was quite literally life-saving, as this treatment has been shown to reduce iron-

related morbidity. However, DFO’s short plasma half-life (about 1 hr) and poor oral bioavailability

(less than 2%) necessitate slow subcutaneous or intravenous infusions with a pump. This procedure

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is very demanding for patients and compliance is poor (less than 70%), even though compliance has

been shown to be absolutely crucial to obtain the full benefit offered by iron chelation (1, 2).

Research aimed at identifying new iron-chelating drugs with improved pharmacokinetic

characteristics and associated compliance, identified deferasirox (Exjade®, ICL670; Novartis Farma

S.p.A.), a once-daily oral iron chelator, which has been designed to treat chronic iron overload in

patients with transfusion-dependent anemias. Because of its convenient oral administration, DFX is

likely also to encourage compliance.

As reported by Waldmeier (3), deferasirox (DFX) showed biopharmaceutical, pharmacokinetic, and

metabolic properties that seem to be favourable for the intended purpose of chelation and

elimination of iron from the body; nevertheless, it can cause digestive disturbances (nausea, stomach

pain or diarrhoea) (4), or cause the patient to change the drug intake time to the evening (5).

Furthermore preparation of the solution from the dispersible tablets is particularly laborious. All

these factors can compromise patient adherence to therapy.

When DFX is administered orally to rats, at least 75% of the dose is absorbed but the bioavailability is

only 26% [3]. This difference between absorption and bioavailability after oral administration is

probably due to hepatic first-pass elimination (6).

The human pharmacokinetic parameters of DFX show a high intra- and inter-individual variability

that may affect the therapeutic response. In a clinical study in healthy volunteers, gastrointestinal

absorption was 70% (3); a median tmax value of 1–4 hours was observed in humans dosed with a

suspension formulation (3, 7). When taken at or close to meal-times, the type of food, its caloric

content and fat content (as well as the timing of the meal with respect to DFX administration) may all

influence its bioavailability and also increase the intra-subject variability of absorption. Therefore,

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patients are recommended to administer DFX at least 30 minutes before a meal for optimum effect

(5).

The DFX and the iron complex are eliminated from the blood through the liver, largely in the bile.

With a mean elimination half-life (t1/2) of 8–16 hours, DFX plasma levels are maintained over 24

hours with once-daily administration (5, 7), but the inter-individual variability of t1/2 is large and

cannot always be estimated. Excretion of DFX and its metabolites occurs mainly within the first 24

hours and is complete within 7 days. The major DFX metabolic pathways are via direct

glucuronidation and conjugation of the hydroxylated metabolites with glucuronic acid and/or sulfate.

In the gut, the acyl glucuronide is unstable and undergoes hydrolysis, probably caused by microbial

glucuronidases, back to DFX, which leads to some enterohepatic recirculation (3). The presence of

large amounts of DFX in the intestine explains the observed reabsorption and enterohepatic

recirculation and the corresponding pharmacokinetics (5).

The DFX glucuronidation is under the control of UDP-glucuronosyltransferase 1A subfamily (UGT1A1

and, to a lesser extent, UGT1A3) (3, 6, 8); Gilbert Syndrome, a mild inherited form of

hyperbilirubinemia, that occurs in 15% of the population, is associated with a UGT1A1 gene

polymorphism. A homozygous insertion of TA pairs (genotype UGT1A1*28/*28) results in a decrease

in bilirubin glucuronidation activity and therefore leads to an increase in the level of unconjugated

bilirubin, although the precise role of the polymorphism is still not completely understood. The

UGT1A1 polymorphism has emerged as an important element in drug tolerance and could increase

the risk of toxicity of drugs metabolized via glucuronidation.

Dosing recommendations for most widely used drugs do not take into account adjustments for

individual characteristics. Knowledge of the gender and physical characteristics of a patient can be

essential for the optimisation of drug therapy, since these can substantially affect the drug’s

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pharmacokinetic parameters and clinical effectiveness (9, 10). Limited preliminary results suggest the

possible influence of gender on the pharmacokinetics of DFX (11).

We report our preliminary experience on the potential usefulness of measurements of DFX plasma

concentration to optimize the drug’s dosage, on the basis of gender and physical characteristics;

individual patient-tailored dosing of DFX should help to improve the efficacy of iron chelation and to

reduce dose-dependent drug toxicity.

We have also evaluated the presence or absence of Gilbert Syndrome in patients, in order to assess

its effect on the efficiency of the glucuronidation metabolic pathway for DFX.

Patients and Methods

Patients

Patients of both sexes aged between 5 and 82 years with transfusion-dependent anemias

(thalassemia, myelodysplastic syndrome, or microdrepanocytosis), and who had been receiving daily

standard treatment with DFX for at least one year, were consecutively enrolled. Patients with any

neurological or psychiatric condition, with unstable or clinically significant cardiovascular disease,

with liver function tests (AST, ALT, bilirubin) ≥ 2 times the upper limit of normal (ULN), and with renal

impairment (creatinine clearance ≤ 30 ml/min according to Cockroft & Gault formula) were excluded

from the study. A signed informed consent was obtained before enrolment. The study was approved

by E.O. Ospedali Galliera Ethics Committee.

All patients received a single oral dose of DFX ranging from 8 to 42 mg/kg/day for at least three

months, and transfusion therapy once every 3–4 weeks. Liver, kidney and heart function and serum

ferritin concentrations were monitored every 4–6 weeks. At the screening visit, age, body weight,

height, and body mass index (BMI), were recorded and lean body mass (LBM) was estimated from

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height and weight using the method of James (12). All patients were genotyped for the UGT1A1*28

gene polymorphism.

Only one blood sample per patient was collected in order not to change current clinical practice or

disrupt the patient’s lifestyle. Blood samples for measuring DFX plasma concentrations (CDFX) were

collected during a routine monthly visit, at the steady state, after the last DFX administration. For

each patient, an aliquot of 4 mL of blood was drawn into heparinized tubes; blood samples were

centrifuged at 1000g for 10 minutes and the resulting plasma was frozen and stored at -20°C until

analysis.

Chemicals and supplies

Deferasirox was kindly provided by Novartis Farma S.p.A., all reagents were of HPLC-grade and were

purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich. The filtration system was obtained

from Millipore S.p.A. (Milano, Italy). A KromaSystem 2000 HPLC system consisting of a 325 pump

system, a 535 UV detector and signal integration software (BIO-TEK Instruments s.r.l. Milano Italy)

was used. Samples were analyzed on a 150 x 4.6 mm I.D. Alltima C18 column (Alltech Italia, s.r.l.,

Milano Italy) and a guard column (Alltech Italia, s.r.l., Milano Italy). Fresh human plasma samples

were obtained from healthy volunteers for standard samples.

DFX determination - Analytical procedure

Deferasirox plasma concentrations (CDFX) was determined with a validated HPLC assay previously

described by Rouan et al. Clinical samples, drug-free plasma and calibration standards were

extracted using this method (13). An aliquot of each extracted sample (100 µl) was injected onto the

HPLC column and eluted with a mobile phase (at pH 7) consisting of 0.05 M di-sodium hydrogen

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phosphate and 0.01 M tetrabutylammonium hydrogen sulfate–acetonitrile–methanol (42:12:46,

v/v/v) at a flow rate of 1.3 ml/min at room temperature. The UV detector was set at 295 nm (ABS

0.1, RT 0.1); the run lasted for 15 minutes in total.

Calibration samples were prepared in pooled samples of blank human plasma and were prepared at

seven different concentrations ranging from 1.25 to 60 μg/ml. The results obtained from the analysis

of the calibration points were examined by linear regression. In order to assess whether a calibration

point could be accepted, it was back-calculated on the basis of the equation of the corresponding

calibration curve; a calibration curve was rejected if more than two concentrations or two adjacent

concentrations deviated more than 20% from the nominal value for the LLOQ (low limit of

quantification) and by more than 15% for the other concentrations (outliers). The calibration curves

of peak areas vs. concentrations of DFX were linear from 1.25 to 60 μg/ml, giving a correlation

coefficient r2 = 0.999. The results, as far as precision and accuracy are concerned, are derived from

the measured concentrations of the validation samples and were acceptable according to

Washington criteria (14).

Statistical analysis

The main endpoint variable considered was the level of CDFX. Summary descriptive statistics included

number (percentage) or rate of subjects for categorical data, mean ± standard deviation (SD) or

median and interquartile range (IQR) for continuous data. To visualize correlations between CDFX

(using the ratio CDFX/dose to adjust for DFX dose) and other factors we used scatterplots with fitted

linear regression lines; to test bivariate correlations, we calculated Spearman's rank correlation

coefficients and p for significance (figure 1 and figure 2). A linear regression model was built with CDFX

at response variable and age, gender, lean body mass, UGT1A1*28 gene polymorphism mutation

status, DFX dose, time from last drug intake to blood sampling and ferritin levels within 6 months

before as explanatory variable (table 2); interaction effects were tested among the factors

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considered. A nonparametric K-sample test on the equality of medians and a nonparametric test for

trend across ordered groups (15) was used to compare CDFX over categories of UGT1A1*28 gene

polymorphism mutation status (figure 3). Two-tailed P value of 0.05 were adopted to define nominal

statistical significance; analyses has been conducted using STATA (version 13; StataCorp., College

Station, TX, USA).

Results

Eighty patients were consecutively enrolled and all successfully completed the study. Table 1

summarizes their demographic data, physical characteristics and laboratory test results. The median

total daily dose of DFX administered was 1500 mg, 25.8 mg/Kg/day (range 8-42 mg/kg/day), median

serum ferritin value in the 6 months before study initiation was 1100 ng/mL (interquartile range 717–

1916 ng/mL). With regard to presence of UGT1A1*28 gene polymorphism, in our cohort, about 40%

of all patients had the wild type allele, while 45% were heterozygous and 16% were homozygous.

Time from last drug intake to blood sampling followed a multimodal distribution, with a median

value of 3 hours (IQR: 1.4-14.0), with 3 main peaks at 2-4, 10-12 and 22-24 hours, as it is easily

deductible from figure 1, panel A. DFX median plasma concentration level was 17.1μg/mL (IQR: 8.8-

38.0): four patients (5%) were below LLOQ (equal to zero).

In figure 1 we show the scatterplots of CDFX/dose ratio vs. time from last drug intake to blood

sampling (panel A) and vs. age (panel B): both the variables were inversely associated with CDFX/dose

ratio (Spearman's rho=-0.25, p=0.03 and -0.18, p=0.1, respectively).

Concerning the association between plasma CDFX/dose ratio and ferritin blood concentration, and

between CDFX/dose ratio and lean body mass (LBM), scatter plots and fitted lines from univariate

linear regression are shown in Figure 2 (panel A and B, respectively). Both ferritin levels and LBM

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appear to inversely correlate with DFX concentration levels, but the association was significant only

for ferritin (Spearman's rho=-0.36, p=0.002 and =-0.11, p=0.3, respectively).

No statistical correlation was detectable between CDFX and DFX dose (Spearman's rho =-0.06, p=0.6;

scatterplot not shown).

Multivariate linear regression model results are shown in table 2. The only two factors significantly,

independently and inversely associated with CDFX levels were time from last drug intake to blood

sampling and ferritin levels in the 6 months before study initiation (both p=0.006). There was a trend

maintain a consistent tense between age and CDFX, even if the statistical significance was not reached

when adjusted for the other factors (p=0.1). Other non significant factors in the model were LBM

(p=0.6), UGT1A1*28 gene polymorphism (p=0.9), gender (p=0.8), DFX dose (p=0.3).

To be noted, is the presence of an interaction between LBM (categorized as below and above the

median, 41.4 kg) and UGT1A1*28 gene polymorphism (three categories), which is significant (p=0.05)

in an univariate linear regression analysis, but remain close to the statistical significance (p=0.09)

adjusting for all other factors. We show this effect modification in figure 3, where a significant

inverse correlation (p=0.03) is evident between CDFX and UGT1A1*28 gene polymorphism, only in

patients with levels of LBM below the median.

Discussion

The goal of iron chelation treatment in patients with iron overload is to induce a negative iron

balance through the removal of excess iron deposited in organs. Reduction of total body iron is highly

dependent upon patient-specific factors including compliance, duration of iron overload, transfusion

burden and the specific properties of each chelator. There is still a need to find safe iron chelators,

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which are able to remove iron from all tissues and which are available in formulations that ensure

maximum patient compliance. The human pharmacokinetic parameters of DFX suggest a high intra-

and inter-individual variability that may affect the therapeutic response and that could lead to

insufficient chelation or increased toxicity; some patients, particularly those with severe iron

overload, fail to respond adequately to DFX at the therapeutic doses, so various strategies have been

investigated to improve iron chelation in patients poorly responsive to DFX. In order to optimize the

drug’s efficacy and/or to monitor dose dependent adverse drug effects, it should be useful to

determine circulating levels of DFX.

Based on the present study results, DFX plasma concentration measured at different sampling times

showed a relatively high variability (interquartile range: 9-38 µg/mL) and is significantly associated

with the time from last drug intake to blood sampling (p=0.006, adjusting for all other factors

considered, table 2). Conversely, there was no linear correlation between CDFX and dose (p=0.3),

substantiating the prediction of a pharmacokinetic inter-individual variability and the observations of

several studies (11, 16). A question is whether no correlation mainly resulted from the inter-subject

pharmacokinetic variability, or also from poor adherence to treatment (i.e. incomplete dosing). Both

inter-subject pharmacokinetic variability and poor compliance have been widely demonstrated; in

our study other variables were taken into account that could affect the plasma concentrations of

DFX, such as gender and age; with regard to gender, in contrast to other authors (11), our results

seem to show a lack of influence of gender on the plasma concentrations, while we found a trend

toward an inverse association between CDFX and age (p=0.1). With regards to compliance, our

evaluation demonstrated DFX plasma concentrations below the quantification threshold (LLOQ) in

four patients (5%), although patients were being treated with therapeutic doses of DFX. All these

factors are a first step in demonstrating the need to monitor DFX plasma concentrations. The strong

inverse association between CDFX and ferritin concentrations (p=0.006) suggests that a higher plasma

DFX concentration could be associated with greater chelation efficacy (17). Since a correlation

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between dose and DFX plasma concentration was not demonstrated, it seems useful to monitor the

concentrations to optimize and determine the most appropriate dose for each patient.

A further interesting result emerged from the analysis of genetic characteristics of patients. Evidence

in the literature suggests that a reduced transcription of the gene coding for UGT1A1, a situation

which is characteristic of Gilbert's Syndrome, may result in a reduction in enzyme activity of up to

70% in subjects with the allelic variant UGT1A1*28 (18-20). However, a mild reduction in the enzyme

does not always result in a full manifestation of Gilbert's Syndrome, which is, in any case, often

asymptomatic and neither diagnosed nor treated (19). Some recent studies have documented the

presence of other allelic variants of UGT associated with reduced enzyme activity (18-21). Although

these enzyme defects do not lead to clinically significant liver injury, they seem to play an important

role in regulating the metabolism of some drugs (18, 22, 23). It is therefore possible to hypothesize

that the presence of particular haplotypes of UGT could have profound effects on DFX disposition

and excretion.

In our study we tested the association between UGT1A1 polymorphisms and CDFX: we did not find any

significant association in the multivariate analysis (p=0.9, table 2), and, although a stratified analysis

of the correlation by mutation status was not feasible due to the low number of samples (lack of

statistical power), we found that LBM (categorized as below and above median value) was a

borderline significant effect modifier (p for interaction: 0.05, figure 3) of the relationship between

UGT1A1 polymorphisms and CDFX.

One hypothesis to justify this finding could be that, as a result of differences in percentage body fat,

lipophilic agents (i.e.DFX) may have a relatively greater volume of distribution (Vd), and water-

soluble compounds (i.e. glucuronate metabolite of DFX) a relatively lower Vd in subject with low

value of LBM. Therefore in subjects with low LBM the presence of UGT1A1 polymorphisms could

generate the lowest DFX plasma concentrations due to a higher distribution on body fat. The

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expected analogous but inverse association (i.e. higher CDFX in UGT1A1 mutated patients with LBM

above the median value) was not observed in patients with LBM above the median value, probably

also because of the low statistical power.

Conclusions

Therapeutic drug monitoring (TDM) is an essential tool to optimize drug dosage; it is an aid to

rational therapy and has become essential in order to tailor the dosage to the individual. Thalassemic

patients might benefit from pharmacologic drug monitoring; measuring the plasma concentration of

DFX may be helpful because low concentrations could reflect either poor compliance or under-

treatment; individual patient-tailored dosing of DFX should help to improve iron-chelation efficacy

and to reduce dose-dependent drug toxicity. This study presented a simple, selective, sensitive, and

convenient method, potentially applicable to routine management of thalassemic patients; the

ruggedness of the analytical procedure ensures the validity and reproducibility of the assay during

standard clinical practice.The individual characteristics of patients could affect pharmacokinetic

parameters and clinical effectiveness of several drugs; for most widely used drugs, dosing

recommendations in adults do not take into account adjustment to individual characteristics. As

suggested by the European Medicines Agency (EMA) and the Food and Drug Administration Office of

Women's Health (FDA-OWH), which both emphasize the need to assess how demographic variables

influence the dose-response relationship defining aspects of the safety and efficacy of drugs (9, 10),

analyses of differences between the genders can provide useful information for possible use in

individualized therapy. Therefore the genetic and physical characteristics should be taken into

consideration during the optimization of drug therapy even if could be hardly feasible in the routine

clinical practice.

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Acknowledgements

The study did not provide any source of funding by the Sponsor.

Authorship Contributions:

Participated in research design: F Mattiolia, V Marinia, C Fucilea, L Robbianoa, S Perrottab, A Rosac,

Antonietta Martellia, G L Fornic

Conducted experiments: V Marini, C Fucile, G Milano

Performed data analysis: M Puntoni, F Mattioli, L Robbiano, A Rosa

Wrote or contributed to the writing of the manuscript: F Mattioli, M Puntoni, C. Fucile,

GL Forni

Conflict of interests: All authors declare that there are no conflicts of interest.

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16. Chauzit E, Bouchet S, Micheau M, et al. A method to measure deferasirox in plasma using HPLC

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Figures captions:

Figure 1. Scatterplots of CDFX/dose ratio vs. time from last drug intake to blood sampling (panel A) and vs.

age (panel B). To visualize correlations, parametric linear regression lines were drawn.

Figure 2. Scatterplots of CDFX/dose ratio vs. ferritin levels in the last 6 months (panel A) and vs. LBM values

(panel B). To visualize correlations, parametric linear regression lines were drawn.

Figure 3. Boxplot of CDFX and mutation for Gilbert’s syndrome, by LBM (median value).

Tables captions:

Table 1. Main characteristics of patients (n=80) undergoing measurement of plasma DFX

concentration.

Table 2. Multivariate linear regression model predicting CDFX with respect to patient characteristics.

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Table 1. Main characteristics of patients (n=80) undergoing measurement of plasma DFX concentration.

Age, yrs (mean ± SD) 31 ± 17

Sex, n (%)

Male 24 (30)

Female 56 (70)

Diagnosis

Beta thalassemia major or intermedia 71 (89)

Myelodysplastic syndrome 6 (7)

Microdrepanocytosis 3 (4)

UGT1A1*28 gene polymorphism (Gilbert Syndrome), n (%)

Wild type 31 (39)

Heterozygous 36 (45)

Homozygous 13 (16)

BMI, kg/m2 (mean ± SD) 22.5 ± 4.0

LBM, kg (mean ± SD) 40.7 ± 9.2

Ferritin levels 6 months before study initiation, ng/ml, (median, IQR) 1100 (717-1916)

DFX dose, mg/kg/day (median, IQR) 25.8 (20.0-32.6)

Time from last drug intake to blood sampling, hours (median, IQR) 3.0 (1.4-14.0)

DFX concentration (CDFX), µg/mL (median, IQR) 17.1 (8.8-38.0)

n: number of observations; SD: standard deviation; IQR: interquartile range (25th-75th percentile); BMI: body mass index; LBM: lean body mass; DFX: deferasirox.

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Figure 1. Scatterplots of CDFX/dose ratio vs. time from last drug intake to blood sampling (panel A) and vs. age

(panel B). To visualize correlations, parametric linear regression lines were drawn.

A

0

2

4

6

C DFX

/dos

e ra

tio

0 10 20 30Time from last drug intake to blood sampling (hours)

Spearman's rho=-0.25, p=0.03

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B

Figure 2. Scatterplots of CDFX/dose ratio vs. ferritin levels in the last 6 months (panel A) and vs. LBM

values (panel B). To visualize correlations, parametric linear regression lines were drawn.

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2

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6 C D

FX/d

ose

ratio

0 20 40 60 80 Age (years)


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B

0

2

4

6

C DFX

/dos

e ra

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10 20 30 40 50 60 Lean Body Mass (Kg)


0

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6 C D

FX/d

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ratio

0 2000 4000 6000 8000 Ferritin levels 6 months before study initiation (ng/mL)


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Table 2. Multivariate linear regression model predicting CDFX with respect to patient characteristics.

Predictor β* 95%CI p

Age -0.2 -0.5 ÷ 0.03 0.1

Time from last drug intake to blood sampling

-0.7 -1.1 ÷ -0.2 0.006

Ferritin levels (6 months before) -0.005 -0.009 ÷ -0.002 0.006

Others not significant factors included in the model were: lean body mass (p=0.6), UGT1A1*28 gene polymorphism (p=0.9), gender (p=0.8), DFX dose (p=0.3). *β is the regression coefficients of standardized data coming from the model.

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Figure 3. Boxplot of CDFX and mutation for Gilbert’s syndrome, by LBM (median value).

The bottom and top of the boxes are the first and third quartiles, the band inside the box is the median, and the ends of the whiskers represent the 9th and the 91st percentiles. Nonparametric K-sample test on the equality of medians of CDFX over UGT1A1*28 gene polymorphism categories: p=0.4 for LBM <41.4kg and p=0.8 for LBM ≥41.4kg.

Notes: *=test for the interaction between LBM* UGT1A1*28 gene polymorphism from the linear regression model on CDFX.

0

20

40

60

80

100

Wild-type

( 15)

Heterozygous

( 21)

Homozygous Wild-type

( 16)

Heterozygous

( 15)

Homozygous

( 9)UGT1A1*28 gene polymorphism (Gilbert Syndrome)

Lean Body Mass <41.4 kg (median) Lean Body Mass ≥41.4 kg (median)

C DFX

(µg/

mL)

P for interaction = 0.05*

P trend=0.03

determination of deferasirox plasma concentrations: do gender, physical and genetic differences...

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