Influence of GST gene polymorphisms on busulfan ... - Nature

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ORIGINAL ARTICLE

Influence of GST gene polymorphisms on busulfan pharmacokinetics in children M Ansari1,2, J-F Lauzon-Joset1, M-F Vachon1, M Duval1,3,4, Y Theóret1,3,5, MA Champagne1,3,4 and M Krajinovic1,3,4,5 1 Charles-Bruneau Cancer Center, CHU Sainte-Justine Research Center, Montreal, Quebec, Canada; 2Department of Pediatrics, Geneva University Hospital, Geneva, Switzerland; 3Clinical Pharmacology Unit, CHU Sainte Justine, Montreal, Quebec, Canada; 4 Department of Pediatrics, University of Montreal, Montreal, Quebec, Canada and 5Department of Pharmacology, University of Montreal, Montreal, Quebec, Canada

Busulfan (BU) is a key compound in conditioning myeloablative regimens for children undergoing hematopoietic stem cell transplantation (HSCT). There are wide interindividual differences in BU pharmacokinetics, which increase the risk of veno-occlusive disease, graft rejection and disease relapse. As BU is mainly metabolized by glutathione S-transferase (GST), it is hypothesized that functional polymorphisms in GST genes may explain in part the variability in BU pharmacokinetics. We analyzed polymorphisms in GSTA1 (C-69T, A-513G, G-631T, C-1142G), GSTM1 (deletion) and GSTP1 (A1578G, C2293T) genes in 28 children undergoing HSCT. All patients had individualized dosing based on pharmacokinetics after the first dose of intravenous BU. GSTM1-null individuals had higher drug exposure (PCmax ¼ 0.008; PAUC ¼ 0.003; PCss ¼ 0.02) and lower clearance (PCL ¼ 0.001). Multivariate regression models showed that, other than the drug dose and age, the GSTM1 genotype was the best predictor of first-dose pharmacokinetic variability. GSTM1-null patients also received lower cumulative BU doses (P ¼ 0.02). No association was found between BU exposure and major GSTA1 or GSTP1 gene variants. In children, GSTM1 polymorphism seems to modify BU pharmacokinetics after intravenous drug administration. Bone Marrow Transplantation (2010) 45, 261–267; doi:10.1038/bmt.2009.143; published online 6 July 2009 Keywords: busulfan; hematopoietic stem cell transplantation; glutathione S-transferase; polymorphisms; pharmacogenetics; pharmacokinetics

Introduction Busulfan (BU) is an alkylating agent used in a wide variety of myeloablative conditioning regimens for hematopoietic stem cell transplantation (HSCT). BU has a narrow therapeutic index. High drug exposure leads to increased risk of veno-occlusive disease (VOD), whereas low drug exposure has been associated with a higher risk of disease recurrence and graft failure.1–4 Accordingly, steady-state BU plasma concentration and area under the concentration–time curve (AUC) correlate with the incidence of these clinical events.1–4 Intravenous (IV) administration has consequently gained popularity, especially in children, as IV pharmacokinetics can be more predictive than pharmacokinetics after oral administration.5–7 Nevertheless, important interindividual variability still persists8 and several additional factors contribute to such observations. Metabolism by glutathione S-transferase (GST) is the main route of BU biotransformation. GSTA1 is the predominant GST isoform catalyzing BU conjugation with glutathione, whereas GSTM1 and GSTP1 have 46 and 18%, respectively, of the activity of GSTA1.9 Interindividual variation may be explained, at least in part, by GST polymorphisms. Two GSTP1 variations leading to Ile105Val and Ala114Val amino-acid replacements result in different catalytic activities.10 A significant number of individuals lack GSTM1 activity (B50%) because of the homozygous deletion of the GSTM1 gene.11 Several polymorphisms, shown in some studies to be functional, have been identified in the GSTA1 gene.12–14 Genotyping patients for relevant polymorphisms, even before drug administration, could further individualize BU treatment.

Materials and methods

Correspondence: Dr M Krajinovic, Centre de recherche, CHU SainteJustine, 3175 chemin de la Coˆte-Ste-Catherine, Montreál, Quebec, Canada H3T 1C5. E-mail: [email protected] Received 2 January 2009; revised 14 April 2009; accepted 21 May 2009; published online 6 July 2009

Patients The study comprised 28 children who underwent HSCT at the CHU Sainte-Justine in Montreal between May 2003 and January 2006. The CHU Sainte-Justine Institutional Review Board approved the study and all of the patients and/or their parents provided informed consent.

GST polymorphisms and busulfan pharmacokinetics M Ansari et al

262

Treatment regimen Sixteen15 doses of IV BU (Busulfex, PDL Biopharma, Edison, NJ, USA) were administered from days 9 to 6 before HSCT: the first dose was age dependent (0.8 mg/kg/ dose for infants X3 months and o1 year of age, 1 mg/kg/ dose for children X1 year and o4 years old and 0.8 mg/kg/ dose for children X4 years old) followed by a pharmacokinetically guided dose adjustment, available before administration of the fifth dose of BU. Patients were scheduled to receive a total of 16 doses of BU. After completion of BU treatment, all patients received cyclophosphamide (cumulative dose of 200 mg/kg) over 4 days. Prophylaxis for seizure was initiated 24 h before and continued for at least 24 h after BU exposure with midazolam, lorazepam or phenytoin. Fluconazole was administered as part of the supportive care regimen after the last dose of BU and antiemetics were routinely administered throughout the BU period. VOD was diagnosed according to the criteria of McDonald et al.,16 that is, the occurrence of two of the following events within 30 days of transplantation: unexplained weight gain of 410% from baseline because of fluid accumulation, jaundice, hepatomegaly or upper right quadrant pain of liver origin. Grading acute graft vs host disease (GVHD) is based on quantification of skin rash, serum bilirubin, diarrhea and persistent nausea according to the 1994 Consensus Conference on Acute GVHD Grading.15 Pharmacokinetics Blood samples were collected immediately before the first BU administration and at 15, 30, 60, 120, 180 and 240 min after drug administration. Plasma BU was determined using a modified high-performance liquid chromatography assay.17 Non-compartmental analysis (WinNonlin, version 3.1) was performed to determine the pharmacokinetic parameters and BU concentration at steady state (Css).8 The remaining BU doses were adjusted to achieve Css between 600 and 900 ng/ml. BU exposure (total AUC over 4 days) in all patients was calculated by the sum of the daily extrapolated AUC measurements.18 Genotyping DNA extracted from the pharmacokinetic blood samples was amplified by polymerase chain reaction using 15–25 ng of genomic DNA. The region from þ 1 to 730 of GSTA1 was amplified as one fragment to detect the single nucleotide polymorphisms (SNPs) at positions 69, 513 and 631.12 The regions containing the GSTA1 1142 polymorphism and GSTP1 gene polymorphisms were also amplified according to published methods.10,19 Detection of the presence or deletion of the GSTM1 gene was as described.20 Genotyping for GSTP1 and GSTA1 was performed by allele-specific oligonucleotide (ASO) hybridization.21 ASO probes for GSTA1 polymorphisms at positions 69, 514, 631 and 1142 were ACTTGACC/ TCTTCTTT, GATAAACA/GATAAATA, ACTTTCAG/ TATTTGTT and ACTGAAC/GGAACTGAG, respecBone Marrow Transplantation

tively. ASO probes for GSTP1 polymorphism were as described earlier.22

Statistics The distribution of pharmacokinetic parameters (AUC, maximum plasma concentration, [Cmax], Css and clearance) obtained after the first BU dose was compared for patients with or without the given genotype/haplotype using ANOVA. Given the limited number of individuals, genotypes were considered as dichotomous variants (the presence or absence of a given genotype). Homozygous and heterozygous individuals for minor (less frequent) alleles were pooled together. Individuals with or without the GSTM1 deletion (null genotype) were considered for GSTM1 gene analyses. Exploratory analysis showed a significant correlation between the GSMT1 genotype and BU mg/kg. No such correlation was seen for BU mg/m2 and the drug plasma concentrations were normalized to mg/m2 for univariate analysis. Body surface area (BSA) was calculated according to the Mosteller formula.23 Multiple linear regression models were also constructed to assess the impact of the GSTM1 genotype (significantly associated in univariate analysis) on BU pharmacokinetics. They included age, sex, phenytoin co-administration and BU dose (in mg/kg) as covariates. The same analyses were used to estimate the impact of the genotype on cumulative drug dose. Estimates of haplotype and haplotype pairs were obtained using PHASE software, version 2.0.24 Statistical analyses were performed by SPSS (Chicago, IL, USA), version 13.0.

Results Patient demographics are shown in Table 1. The SNP at position 69 of GSTA1 (C to T) was chosen for analysis because it was previously shown that this SNP is in complete linkage disequilibrium with T-567G, G-52A and A375G substitutions and because it defines haplotypes *A and *B.12 C-1142G and G-631T were included in the analysis, as they were shown to further diversify *A and *B haplotypes into different subtypes.19 An A-513 to G substitution was reported by Guy et al.13 Analysis in our group of patients revealed six haplotypes. Five were previously identified (A1, *A2, *A3, *B1, *B2), whereas a sixth, here defined as *B1a, differed from *B1 by the presence of the G-513 allele. The SNPs analyzed in the GSTA1 gene and derived haplotypes with respective frequencies are given in Table 2. Association analysis with pharmacokinetic parameters revealed no difference for the GSTA1 polymorphisms or major *A/*B haplotypes (data not shown). The difference was seen when correlations between GSTM1 genotypes and dose-normalized (mg/m2) drug concentrations and clearance were analyzed. GSTM1-null individuals (n ¼ 14) had higher drug exposure (AUC, P ¼ 0.003; Cmax, P ¼ 0.008; Css, P ¼ 0.02) and lower clearance (P ¼ 0.001) compared with non-null individuals (Figure 1a).


263 Patient characteristics

Table 1 Age (years)

Sex

Weight (kg)

Diagnosis

0.4 0.5 0.6 0.6 1.5 2.2 2.4 4.4 4.7 4.9 5.5 8.2 8.5 10.4 11.6 11.8 16.3 17.1 5.2 5.7 6.2 8.1 10.4 10.6 16 16.8 17.6 19.8

F F F M F F F F F F F F F F F F F F M M M M M M M M M M

6.2 6.5 8 8 10.3 10.7 12.3 14.8 21 21.5 19 23.2 27.1 50.2 57.5 52.6 52.5 49.2 24.1 23.2 27.9 28.9 29.5 27.3 63.5 65.3 84.5 51.5

FELH FELH ML AML FELH ID MDS MDS SC MDS MDS SC MDS AML SC SC AML AML AML MDS MDS AML AML AML SC AML ALL ALL

Seizure prophylaxis

Css (ng/ml)

AUC (ng/ml/min)

Cumulative dose (mg/kg)

GSTM1

GSTA1 *B

L L L M M M L M L L M L L+P L L L P P M L L P L L L M M P

494 647 631 404 720 603 688 603 497 530 574 325 549 755 632 523 773 690 528 478 617 655 546 414 717 578 671 936

163208.4 224335.5 211441.6 133315.1 243610.6 259783.6 239797.2 206515.3 167148.3 179258.3 171454.9 108767.2 187028.5 261904.7 216713.5 177969.1 267086.6 240978.2 163946.3 165859.1 213482.7 228188.9 182200.5 141151.4 251127.6 198242.5 233872.8 329655.8

15.56 14.85 18.75 14.50 16.75 16.51 16.83 14.46 15.86 15.21 12.42 19.31 15.42 12.75 12.52 16.35 12.80 13.01 17.63 19.31 13.80 13.28 17.83 19.27 12.60 13.94 12.92 10.68

Null Non-null Null Null Null Null Null Null Non-null Null Non-null Non-null Non-null Null Non-null Non-null Null Non-null Non-null Non-null Null Null Null Non-null Non-null Non-null Non-null Null

+ + + + + + + + + + + + + + + + + + + +

VOD

GVHD

+

+ +

+

+

Abbreviations: AML ¼ acute myeloid leukemia; ALL ¼ acute lymphoblastic leukemia; F ¼ female; FELH ¼ familial lymphohistiocytic syndrome; GSTM1 and GSTA1 ¼ glutathione S-transferase (GST) genotype M1 and A1. GSTA1 *B ¼ carriers (+) or not of haplotype *B; GVHD ¼ graft vs host disease (gradeX2); HSCT ¼ hematopoietic stem cell transplantation; ID ¼ immune disease; L ¼ lorazepam; M ¼ male; M ¼ midazolam; MDS ¼ myelodysplastic syndrome; ML ¼ metachromatic leukodystrophy; P ¼ phenytoin; SC ¼ sickle cell disease; VOD ¼ veno-occlusive disease. Data are organized according to age group with various dosing schedules (o1 year, between 1 and 4 years and 44 years). The median age in respective groups was 0.5, 2.2 and 10.4 years. Median first dose received was 0.81, 0.98 and 0.79 mg/kg, and the median cumulative dose was 15.9, 16.7 and 14.8 mg/kg. The median value of the steady state concentration (Css) was 562.5, 688 and 578 ng/ml, and the median value of area under the plasma concentration–time curve (AUC) was 183 075.2, 247 730.5 and 204 407.2 ng/ml/min. The number of related and unrelated donors was 14 in each group. Stem cell source was cord blood in 12 cases and bone marrow in 16 cases.

Table 2

*A1 *A2 *A3 *B1 *B2 *B1a Minor allele

Frequency of GSTA1 polymorphisms and haplotypes 69

513

631

1142

C C C T T T 22 (39.3)

A A A A A G 1(1.8)

T G T G G G 17 (30.4)

C C G G C G 20 (35.7)

Haplotypes 17 16 1 18 3 1

(30.4) (28.6) (1.8) (32.1) (5.3) (1.8)

Haplotype carriers 16 13 1 17 3 1

(57.1) (46.4) (3.6) (60.7) (10.7) (3.6)

Haplotype and minor allele frequency was calculated relative to the total number of chromosomes. Carriers of a given haplotype were calculated relative to the total number of individuals with all haplotype pairs containing the respective haplotype. Numbers and percentages (in parentheses) are given.

Exploratory analysis showed the correlation between the GSTM1 genotype and BU mg/kg, which can confound the genotype effect if the pharmacokinetic parameters are normalized to the first drug dose in mg/kg using univariate analysis. A linear regression model was therefore built to control for this effect as well as for the effect of other covariates on exposure (AUC, Cmax and Css). GSTM1 genotype and first BU dose (mg/kg), along with sex, age and co-administration (or not) of phenytoin for convulsion

prophylaxis, were included in the analysis. Co-administration of phenytoin was considered as a covariate because it was reported to increase BU elimination.25 Final models (Table 3) were obtained by stepwise selection and retained only covariates significantly associated with BU exposure. BU dose, age and GSTM1-null genotype (AUC, P ¼ 0.02, Cmax, P ¼ 0.001 Css, P ¼ 0.004) were significantly associated with drug exposure. The models obtained explained 40–50% of the variability of pharmacokinetic parameters. Bone Marrow Transplantation


264 Cmax ng/mL

AUC ng/mL/min 55

Css ng/mL

Clearance ml/min

40

14000 50

200 180

35

12000

160

45 30

10000

140

40

8000

35

6000

30

120

25

100 20 80 p=0.008

4000

350000 300000 250000 200000 150000 p=0.03

100000 –

+

25

p=0.003

15

1200

1000

1100

900

1000

800

900

700

800

600

700

500

600

400

p=0.02

p=0.001

60

300 250 200 150 100

p=0.002

500 –

300

+

p=0.03 –

+

50

p=0.04 –

+

Figure 1

Pharmacokinetic parameters in all individuals (a) or patients older than 4 years (b) who have or do not have the GSTM1-null genotype. Each individual value is represented by the symbol (J), whereas the line represents the respective mean value. P-values for the difference in indicated parameters between groups with ( þ ) and without () the GSTM1-null genotype, obtained by ANOVA, are indicated on the plots. AUC, area under the plasma concentration–time curve; Cmax, maximum plasma drug concentration; Css, steady state concentration; GSTM1, glutathione S-transferase M1 genotype. AUC, Cmax and Css were normalized to the busulfan mg/m2 and clearance is adjusted to body surface area in Figure 1a or pharmacokinetic parameters were used uncorrected in Figure 1b. (a) Respective mean±standard error of mean (s.e.m) for individuals with and without the GSTM1-null genotype: AUC ¼ 10023±326 vs 8206±536, Cmax ¼ 42.1±1.7 vs 33.2±2.0, Css ¼ 28.9±0.8 vs 24.4±1.7, clearance ¼ 100.1±3.4 vs 127.7±6.7, (b) Respective mean±s.e.m. for individuals with and without the GSTM1-null genotype: AUC ¼ 233537±17884 vs 186481±11364, Cmax ¼ 920.2±45.8 vs 752.8±24.5.0, Css ¼ 676.9±48.3 vs 552±30.9, clearance ¼ 113.7±0.2 vs 165.2±16.5.

Co-administration of phenytoin was not significantly associated with plasma drug concentrations (P40.2) and was not retained in the final model. We used a similar approach to analyze the effect of GSTM1-null genotype on cumulative BU doses including weight, sex, age and administration of phenytoin as covariates. GSTM1-null genotype was associated with lower cumulative BU doses (P ¼ 0.02). Finally, we added a further univariate analysis to assess an association of GSTM1 genotypes and first-dose pharmacokinetics in the subset of patients (n ¼ 21) belonging to the largest age group (44 years of age) that received the same first BU dose. Eight patients were with GSTM1-null genotype. The results remain significant (Pp0.04, Figure 1b) and reflect the same pattern as the results obtained when the whole group of patients was analyzed. Three patients out of 28 (10.7%) developed VOD (Table 1) and only one had GSTM1 null genotype. BUcorrected total AUC was 22 089 vs 11 691 ng/ml/min in Bone Marrow Transplantation

patients with and without VOD, respectively (P ¼ 0.08). Two patients (7.1%) developed GVHD (grade X2, Table 1), and both had GSTM1-null genotype. No correlation between GVHD and BU exposure was seen (P ¼ 0.9) No differences in pharmacokinetics were seen in terms of GSTP1 polymorphisms or haplotypes.

Discussion The introduction of BU as an IV formulation, followed by a dose adjustment based on first-dose pharmacokinetics, reduces the variability of BU exposure among patients, as shown in several studies.7,26–28 Dose targeting based on therapeutic drug monitoring (TDM) also seems to improve event-free survival and survival rates and in some studies VOD-free survival compared with conventional BU treatment.18,29,30 Despite these improvements, inter-patient


265 Table 3 Linear regression models analyzing the relationship of GSTM1-null genotype with (a) busulfan plasma concentrations and (b) cumulative drug doses in the presence of other relevant factors Covariatesa

Cmax (ng/ml) B

b

(a) GSTM1 156.1 Dose (mg/kg) 604.2 Age 12.3 Sex 122.4 Covariatesa

(b) GSTM1 Weight (kg)

AUC0N (ng/ml/min) b

P

B

0.001 0.03 0.003 0.006

36495.1 225747.8 5580.0

Css (ng/ml)

P

Bb

P

0.02 0.02 o0.0001

126.2

0.004

12.4

0.001

Cumulative drug dose (mg) Bb

P

61.4 11.7

0.02 o0.0001

Abbreviations: AUC ¼ area under the plasma concentration–time curve; Cmax ¼ maximum plasma drug concentration; Css ¼ steady state plasma concentration; GSTM1-null, GSTM1 ¼ glutathione S-transferase M1 genotype. a GSTM1 and sex are dichotomous variables, absence of GSTM1 deletion and female sex are respective reference categories. Age, dose and weight are continuous variables. b Beta, unstandardized regression coefficient.

variability still exists and the percentage of patients reaching the target AUC after the first dose remains low.8,18,26,31,32 Several factors have been identified to account for differences such as sex, weight, age or BSA.4,26,33 In this paper, we examined whether the polymorphisms in the GSTA1, GSTM1 and GTP1 genes also contributed to this variability. We found that GSTM1-null individuals had a significantly higher plasma drug concentration and lower clearance compared with non-null individuals as would be expected for subjects lacking GSTM1 activity. GSTM1-null patients also received lower cumulative BU doses. However, many other factors could have contributed to the decrease in dose seen in these patients.26,33 Srivastava et al.34 reported a higher incidence of VOD in GSTM1-null thalassemia patients who did not have BU dose adjustments to achieve the predetermined target level. This finding accords with studies that suggest a relation between VOD and a higher BU exposure.2–4 However, they reported lower drug levels and higher clearance in GSTM1-null individuals, suggesting that VOD may be mediated by glutathione depletion, possibly through higher GSTA1 activity. The study of Bredschneider et al.19 contrasts with this explanation by showing that neither GSTA1 protein expression nor conjugation activity was affected by GSTM1 status in human liver tissue. Two other studies that analyzed the GSTM1 gene did not find any significant correlation between first-dose pharmacokinetic variability and the GSTM1-null genotype.33,35 The difference in the patient population and treatment schedule or other factors contributing to this variability may account for a discrepancy seen across studies. Interestingly, among the patients we analyzed, 50% had Css values below the lower target level limit, similar to other studies.8,18,31 This may

suggest that patients with the non-null GSTM1 genotype could receive a higher first dose. It remains to be seen in larger studies whether any of these genotypes may predict BU-related side effects. Given that TDM is performed in these children, an association with pharmacokinetics variability after the first BU dose does not necessarily reflect an association with disease outcome.35 For example, three patients out of 28 (10.7%) enrolled in this study developed VOD and only one had the GSTM1-null genotype. BU exposure was slightly higher in patients with VOD compared with those without this complication; however, difference did not reach significance (P ¼ 0.08). It seems that many factors are equally important for VOD development, including underlying disease, pre-existing risk for VOD and particularly other drugs administered concomitantly with BU.2,18,36 Recently, a higher incidence of acute GVHD (grade X2) was seen in patients with greater BU exposure.18 In our study group, there were only two such patients; both had GSTM1 null genotype and no correlation was seen with BU exposure (P ¼ 0.9). We did not find any association of haplotype *B with pharmacokinetic variability with the number of patients we analyzed. The absence of such an association in pediatric patients was also reported by Zwaveling et al.,33 whereas others, in contrast, reported higher BU concentration or lower clearance in individuals with GSTA1 *B.35,37 Higher drug concentration is in accordance with lower enzyme activity reported for this haplotype.12,14 The functional impact of GSTA1 gene variations still remains to be clarified, as Bredschneider et al.19 showed no association of GSTA1 protein expression and conjugation activity with GSTA1 *B or GSTA1 polymorphisms. It was not possible to estimate the contribution of haplotype subtypes or homozygosity for minor alleles of GSTA1 polymorphisms owing to the small number of patients. In keeping with previous results,33,35 no association was found between GSTP1 variants and BU pharmacokinetics.

Conclusion In conclusion, we found that the GSTM1-null genotype contributed to pharmacokinetic variability after the first BU dose. Further and larger studies are needed to replicate this finding and to address the role of GSTA1 polymorphisms in both association and functional studies. Different population-based modeling in combination with the identification of covariates that account for the pharmacokinetic and pharmacodynamic variability of BU may allow us to better estimate first drug dose and efficacy/safety profiles in pediatric patients.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements We are thankful to all the patients and their parents who consented to participate in this HSCT-related genetics study. Bone Marrow Transplantation


266

This work was supported by grants from PDL Biopharma, the Fondation Te´le´maque and Fondation du centre de cance´rologie Charles-Bruneau. MK is a scholar of the Fonds de la Recherche en Sante´ du Que´bec. MA is a scholar of the Fondation Te´le´maque and the Fondation Charles-Bruneau.

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