Introduction

Sickle cell anemia (SCA) is a severe hematological disease caused by the recessive autosomal inheritance of the beta (β) S globin gene that affects approximately 300,000 to 400,000 newborns worldwide each year [1-3]. SCA individuals present clinical heterogeneity, and those with a severe clinical profile, i.e, recurrent vaso-occlusion crises and acute chest syndrome, the occurrence of osteonecrosis and retinopathy, as well as stroke risk, are commonly indicated to use hydroxyurea (HU). HU (CAS Registry Number, 127-07-1) is an antineoplastic drug approved by the U.S. Food and Drug Administration to treat SCA due to its anti-sickling potential [4,5]. However, inter-individual variations in the therapeutic response to HU are frequently observed, and this may be attributed to several factors, including variations in the metabolic profile of the individuals [6]. Despite its broad indication and usage, the HU metabolism has yet to be completely elucidated. Evidence has shown that the first step of the HU conversion into nitric oxide (NO) is mediated by catalase [7]. Moreover, Juul et al. [8] demonstrated that HU effects depend on its direct target for catalase and suggested that it could act as a catalase-activated prodrug. Catalase (CAT; EC 1.11.1.6) is an endogenous antioxidant enzyme crucial in neutralizing reactive oxygen species. This enzyme, encoded by the CAT gene, located on chromosome 11 (11p13.31), is most expressed in the liver, kidney, and erythrocytes [9,10]. Although controversial, the polymorphism -21A>T (rs7943316) in the promoter region of the CAT gene has been linked to changes in CAT transcription and enzymatic activity [1013]. The present study investigated the effects of the CAT -21A>T variant on pharmacokinetic (PK) parameters in SCA children undergoing HU therapy.

Material and Methods

Subjects and ethical aspects

We included 12 SCA children (HbSS) undergoing HU therapy, all seen at the University Hospital Professor Edgard Santos (HUPES) outpatient clinic in Salvador, Brazil. All children reported regular use of folic acid and were in disease steady-state, defined as the absence of acute crisis and no blood transfusion in the three months before blood collection. The exclusion criteria were chronic transfusion therapy, active infection, or inflammatory diseases. This research protocol received approval from the Institutional Review Board of HUPES (protocol number: 2.823.416), and individuals or their legal guardians provided a signed term of informed consent before enrollment in the study. The study protocol was performed in accordance with the Helsinki Declaration of 1975 and its later amendments.

Pharmacokinetic and laboratory parameters

Blood samples were collected by venipuncture in the morning, after 12h of fasting, under standardized conditions, before HU administration (T0), and 45, 90, 120, 150, 180, 240, 360, and 480 minutes later, in tubes containing heparin for PK assay. The quantitative measurement of the plasma HU concentration was performed by high-performance liquid chromatography with a mass spectrometry detector (HPLC/MS). The PK profile of each patient was obtained from non-compartmental analysis using PKanalix® 2021R1 software (Lixoft, France). PK parameters such as the area under the concentration-time curve from time zero to last time or infinity (AUC0–t or AUC0-inf), apparent elimination rate constant (Lambda), mean residence time (MRT), apparent total clearance (CL/F), and apparent volume of distribution (V/F), were determined. At T0, blood samples were collected in tubes without anticoagulant or with anticoagulant EDTA for laboratory parameters analysis. Hematological, biochemical, and inflammatory parameters were assessed using a Beckman Coulter LH 780 Hematology Analyzer (Beckman Coulter, Brea, California, USA), an A25 spectrophotometer autoanalyzer (Biosystems SA, Barcelona, Spain), and an Access 2 Immunoassay System (Beckman Coulter, Fullerton, CA, USA), respectively.

Molecular analysis

CAT -21A>T variant was investigated from genomic DNA using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique [10]. Moreover, the genetic modifier, β ^S haplotypes, was also investigated by PCR-RFLP [14].

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 and SPSS 17.0, with p<0.05 considered statistically significant. The frequencies of the categorical variables were calculated, and to test whether CAT -21A>T variant was in HardyWeinberg equilibrium, a goodness-of-fit χ² was used. Association analyses between PK parameters and CAT -21A>T variant were performed using codominant and recessive genetic models. In the codominant genetic model, ANOVA or Kruskal Wallis was used to compare mean values between 03 genotype groups (wild type vs. heterozygote vs. variant). Regarding the recessive genetic model, we performed an unpaired t-test or Mann-Whitney U test to compare mean values between 02 groups (wild type and heterozygote vs. variant). Moreover, multivariate linear regression analyses were performed to confirm the influence of the variant on PK parameters in the presence of confounding variables. Data were expressed as mean ± standard deviation, median (minimummaximum), or number or frequency where appropriate.

Results

Socio-demographic, laboratory, and pharmacokinetic data

The median age of the children was 13 years (range: 8-17 years), and four (33.33%) of them were female. The HU dosage ranged between 15.94 and 24.90 mg/kg/day (median: 20.26), and the average duration of HU treatment was 52 months. Table 1 presents laboratory parameters. Data shows increases in fetal hemoglobin (HbF), reticulocytes, MCV, MCH, RDW, iron serum, LDH, AST, and total and indirect bilirubin, as well as reductions in red blood cell count, hemoglobin, and hematocrit when compared to reference values. Table 2 presents estimated HU PK parameters, and Figure 1 shows HU plasma concentration vs. time data after administering oral doses of HU in SCA children. The mean HU oral clearance was 40 ± 16 L/h, while HU Cmax and AUC0-inf were 9998.25 ± 4023.02 ng/mL and 22605.46 ± 10041.57 ng*h/ mL, respectively.

CAT -21A>T and β^S haplotypes frequencies

CAT -21A>T variant was found to be in Hardy-Weinberg Equilibrium (χ ² = 0.34, df = 1, p = 0.545). Two (16.67%) children had the wild type (CAT -21AA) genotype, while 07 (58.33%) and 03 (25.00%) had heterozygote (CAT -21AT) and variant (CAT -21TT) genotypes, respectively (Figures 2A and 2E). β ^S haplotypes allelic distribution is presented in Figure 3.

Association between CAT -21A>T variant and PK parameters and multivariate linear regression analyses

Association analyses were performed to investigate the influence of the CAT -21A>T variant on PK parameters. Using a codominant genetic model, we observed a significant association between variant CAT -21TT genotype and an increase in HU clearance, while in the recessive genetic model this genotype was significantly associated with an increase in HU clearance and a reduction in HU Cmax (Figure 2). Table 3 presents data of the multivariate linear regression. The proposed model significantly predicted Cmax, F (2, 9) = 4.542, p = 0.043. The adjusted R² = 0.392 depicts that this model explains 39.2% of the variance in Cmax, where the variant CAT -21TT genotype was significantly associated with a reduction in Cmax (β = -0.635, p = 0.025). HU clearance was also significantly predicted by the proposed model, F (2, 9) = 7.337, p = 0.013. Moreover, the adjusted R² = 0.535 shows that the model explains 53.5% of the variance in clearance, where the variant CAT -21TT genotype presented a significant association with an increase in clearance (β = 0.768, p = 0.005).

reinforced by the elevated concentrations of hemolytic biomarkers (iron serum, LDH, AST, and total and indirect bilirubin) and corroborates the findings of previous studies [16,17]. Moreover, children had MCV, MCH, and RDW higher than reference values, which probably resulted from HU use, as shown in other studies [18,19]. PK parameters are critical for understanding drugs’ efficacy and safety. In the present study, we observed in the SCA children a mean HU oral clearance (40 L/h) higher than in some reports, ranging from 4.5 to 27.1 L/h [20-25]. In contrast to data from previous studies [25,26], we also observed a lower HU plasma exposure, characterized by lower AUC and Cmax, in the investigated children. These discrepancies between Brazilian children and other study populations may be due to various factors, including sociodemographic and genetic characteristics, and deserve to be better elucidated. Reports demonstrated the crucial role of polymorphisms in genes encoding drug-metabolizing enzymes and drug transporters in therapeutic response [27,28]. In the present study, we focus on catalase, an endogenous antioxidant enzyme associated with HU conversion. The genotypic and allelic frequencies of the CAT -21A>T variant observed in the present study are consistent with our previous findings [28]. This data also corroborates frequencies previously published [10]. Analyses showed that the CAT -21A>T variant is associated with HU PK parameters. Despite the limited sample size, the CAT variant (CAT -21TT) genotype was significantly associated with increased HU clearance and decreased Cmax, using a codominant and recessive genetic model. Furthermore, multivariate linear regression analysis confirmed associations between the variant genotype and the observed alterations in PK HU, considering patient age or body weight as confounding variables. Indeed, previous studies used these variables to test population PK models of HU [22,29]. Dong et al. demonstrated that body weight might be a significant predictor for both maximum elimination rate and apparent volume of distribution [22]. These results suggest that the variant CAT -21T allele in homozygous trends to increase HU clearance decreasing Cmax. Reports regarding the effect of variant -21A>T in CAT expression are controversial [10-13]. Of these studies, only Saify et al. [13] investigated the association between this variant and CAT expression. Taking into account its association with an increase in CAT mRNA and the fact that HU can act as a catalase-activated pro-drug [8], our findings suggest that carriers of the variant -21TT genotype produce higher prodrug levels. This hypothesis correlates with lower HU Cmax and higher clearance observed in individuals with the variant genotype when compared to those with the CAT -21AA or AT genotype. Moreover, the reduced levels of the pro-inflammatory biomarker alpha-1 antitrypsin previously reported in CAT -21 TT carriers [28] suggest that higher exposure to the active metabolite may influence HU response. The main limitation of the present study is the low number of individuals included. However, this does not detract from the results since the CAT -21A>T variant was in HardyWeinberg Equilibrium. Contrarily, the findings arouse interest in continuing the investigations in this line.

Conclusion

Our findings demonstrated the lower HU plasma exposure in Brazilian SCA children and the association of CAT -21A>T with alterations in HU PK parameters, which has never been reported. However, further studies are necessary to validate the lower HU plasma exposure from a large sample and establish a cause-effect relationship between this variant and the functional effects and metabolic profiles of SCA individuals undergoing HU therapy. Whether confirmed, variant CAT -21A>T might be used to propose new pharmacogenetics-guided dosing strategies for HU therapy.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

MSG, ALG, and SCMAY conceived and designed the research. SCMAY, JSSN, LMF, CCG, SPC, RPS, and AFF performed research. NVM, FJA, SCMAY, and JSSN analyzed data. ILM followed-up and indicated patients. MSG provided financial resource. SCMAY wrote the first draft of the manuscript. MSG, ALG, and EVA revised the draft. MSG supervised the study. All authors revised the final version of the manuscript and consented to submission.

Funding

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

(470959/2014- 2 and 405595/2016-6; MSG) and (152408/20207; SCMAY); Programa Inova Fiocruz - Edital Geração de Conhecimento (VPPCB-007-FIO-18-2-66; MSG); and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) (Finance Code 001; SCMAY, SPCV, RPS, and LMF). The sponsors of this study are public or nonprofit organizations that support science in general.

Acknowledgments

The authors would like to thank all of the children and their legal guardians for their kind participation in the present study. We also thank all personnel at the outpatient service of the Hospital Universitário Professor Edgard Santos (HUPES), Núcleo de Ensaios Clínicos da Bahia (NECBA); Departamento de Toxicologia e Análises Clı́nicas, Faculdade de Farmácia (LACTFAR-UFBA); and Serviço de Equivalência Farmacêutica e Farmacocinética (Sefar), Fiocruz. Our special thanks to Prof. Nicolaus Albert Borges Schriefer, Maria São Pedro Bispo dos Santos Trindade, Prof. Carolina Oliveira de Souza, and Prof. Davyson de Lima Moreira.

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