Impact of CYP3A4 rs2242480 polymorphisms on Imatinib HPLC determined trough concentration and response in Chronic Myeloid Leukaemia in Pakistani patients

Introduction

The prognosis of Chronic Myeloid Leukaemia (CML) has changed significantly in the last two decades with the introduction of tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), as the standard treatment. The efficacy of IM is determined by its concentration in the plasma. However, there is a noticeable variability in the response among individuals, and even within the same patient, when blood samples are taken at different times. This variability in drug response could be due to changes in pharmacokinetic processes, along with other factors (Natarajan, Kumar et al. 2019). Single nucleotide polymorphisms (SNPs) or single nucleotide variants (SNVs) may affect the plasma drug concentration and hence the therapeutic response. Genes that encode transporters and metabolizing enzymes are of special interest as SNP for these enzymes may explain the resistance developed against Imatinib Mesylate in CML initially or during treatment. The cytochrome P450 enzyme system extensively metabolizes imatinib in pharmacokinetic processes (Pena, Muriel et al. 2020). The main enzyme involved in vivo metabolism is CYP3A4. Multiple studies have indicated that individuals with minor alleles of SNP rs2242480 of CYP3A4 may have lower plasma concentrations (Pena, Muriel et al. 2020). Considering this, a decreased response to treatment in individuals with minor allele frequencies could be anticipated (Ullah, Ali et al. 2022). The predicted response can lead to the adoption of strategies like therapeutic drug monitoring for Imatinib with individualized adjustment of doses to achieve the therapeutic goal during the early stages of treatment (García-Ferrer, Wojnicz et al. 2019).

This study aimed to explore the impact of minor allele frequencies of rs2242480 on the response to imatinib and plasma trough concentration with the effects of different genetic models on the outcome of treatment.

Methods

Results

In total, 120 patients were included in this study. Of these, 14 were excluded because they did not meet the inclusion criteria. complete Haematological response was considered in responders when the WBC count was less than 10×10³/μL, basophils were less than 6% with no myelocytes, promyelocytes, or myeloblasts in the differential leukocyte count, and platelets were less than 450×10³/μL with non-palpable spleen (Farooq, Khan et al. 2019). 54.7% of patients were male, while 45.3% patients were female. A significant association was found between sex and increased response in females as compared to males (64% vs. 36%). The imatinib trough concentration in males was lower than that in females (875 vs. 1151ng/ml). (Of the total sample size, 11 patients were above 60 years of age). There was an increased percentage of responders with increasing age and imatinib levels of 625 ng/ml in group 1 compared to 1240 ng/ml in group 4. None of the observed genotype frequencies were significantly different from the expected value (p > 0.05), illustrating that these frequencies follow Hardy-Weinberg equilibrium (Table 1).

Of the 106 sample sizes, 49 patients had a CC variant of CY3A4 rs2242480, 38 patients had a CT variant, and 19 patients had a minor TT allele. Plasma HPLC levels of trough concentration of imatinib were calculated in all three variants with the highest trough levels of 1298 ng/ml in patients with the CC variant, while CT levels were 873ng/ml and TT levels were 489 ng/ml (Figure 1). Analysis of variance was used to determine the difference between the polymorphic variants, which was found to be significant (p-value 0.000) (Table 1). The p value between the wild homozygous and heterozygous individuals was 0.015. Similarly, the p-value was again found to be significant (0.003) between heterozygous (CT) and mutant homozygous (TT).

Figure 1. Plasma trough concentration of Imatinib in genotypes of rs2242480 in CML patients determined through HPLC.

Abbreviations: HPLC: high-Performance Liquid Chromatography; CML: Chronic Myeloid Leukaemia.

CYP3A4 rs2242480 polymorphism in genetic models with impact on Imatinib concentration in non-responders and responders.

Analyzing responders and non-responders, rs2242480, minor allele TT was associated with decreased response to imatinib as a significant p-value in codominant (p-value 0.005), dominant (p value0.002), recessive (p-value 0.02), and additive models (p-value 0.000), whereas in the over-dominant model, the minor allele TT caused a decrease in response with genotypes CC/TT with a p-value of 0.199 (Table 2).

Discussion

Drug transporters, including entry and efflux transporters, along with enzymes involved in imatinib metabolism, may have a substantial influence on therapeutic response. The pharmacokinetic processes of imatinib depend on the efficiency of a protein involved in drug absorption and disposition. After oral administration of IM, it is mainly metabolized by the Cytochrome P450 enzyme system by CYP3A4 and CYP3A5 enzymes. The entry transporters involved are from the ABC subfamily. Organic cationic transporter 1(OCT), encoded by SLC22A1, cannot be ignored because of its importance in the uptake and extrusion of small toxic by-products produced endogenously. Imatinib has changed the course of this cancer, but it is now widely accepted that resistance to this drug is emerging. In this study, we explored the effect of genetic polymorphisms on imatinib trough concentration determined using HPLC and its association with the outcome of treatment in responders and non-responders. This genetic polymorphism was identified against CYP3A4 rs2242480, and data were collected against each allelic form and its association with response. Sex and age associations were also assessed. Imatinib determined by HPLC was also analyzed in both sexes, in different age groups, and against each allelic form.

During the study period, from December 2020 to February 2022, blood samples were collected after steady-state concentration was achieved, and it was revealed that HPLC trough levels of imatinib were higher in the elderly than in the younger age groups, with a highly significant p-value (0.01) among groups. The trough levels of imatinib were 625 mg/ml, 780 mg/ml, 801 mg/ml, and 1240 mg/ml from group 1 to group 4, respectively, showing a gradual increase in the concentration of imatinib with increasing age. The potential mechanism behind this could be attributed to the lower volume of distribution and slower metabolism. This finding may guide treating physicians for therapeutic drug monitoring in both elderly and younger patients for dose adjustments with better treatment outcomes in terms of a decrease in adverse effects and relatively higher doses in young patients to achieve therapeutic concentration (Gao, Wang et al. 2023). Our study was in alignment with the study done in July 2023 by Adattini, Adiwidjaja et al. (2023) which showed increased concentration in the older patient population with earlier achievement of complete molecular response and more adverse effects beyond the therapeutic concentration. Another study conducted in April 2020 by Xia, Chen et al. (2020) concluded that age could be a significant covariate in determining imatinib concentration, which also supports this study for anticipating age-related adverse effects with underlying supra-therapeutic imatinib concentrations. The association of response with age was also determined through X² and it was found to be significant (p value 0.02) with more responders to the treatment in the elderly, which aligns with the effectiveness of Imatinib in CML as determined by Chen, Dong et al. (2020). In terms of response, it was found out that females have a better response as compared to males with responder’s percentage (64% vs 36%) as shown in Table 1 and highly significant association (p value 0.000). The reason for this better outcome could be differences in pharmacokinetic processes, better immune response, or adherence to the treatment, as shown by Breccia, Molica et al. (2018). Imatinib plasma concentration was also higher in females (1151 ng/ml) vs males (875 ng/ml).

Conclusion

There is a potential way to improve the outcomes of CML treatment by considering the relationship between SNPs and low imatinib levels in patients. Despite these challenges, continuous research and collaboration among healthcare experts can help to develop personalized treatment plans. This, in turn, can lead to better safety and effectiveness of Imatinib treatment for patients suffering from chronic myeloid leukaemia.

Ethics and consent

The study protocol was approved by the Islamic International Medical Collegeethical review committee Ref#Riphah/IIMC/ERC/19/0265 dated 25^th July 2019. The study protocol were in accordance with the declaration of Helsinki and guidelines provided by good clinical practice.

Informed written consent was obtained from all participants before the start of the study.