Genetic predictors of morphine concentration: ABCB1 polymorphisms’ importance in palliative oncology

Introduction

Pain management remains a central component of palliative therapy in patients with oncological diseases. Despite existing standardized dosing regimens, morphine is characterized by significant interindividual variability in terms of analgesic efficacy and tolerability profile. One of the leading factors contributing to this variability is the functional activity of transporter proteins involved in regulating the penetration of drug substances across the blood-brain barrier. The ABCB1 gene (also known as MDR1) encodes the membrane transporter P-glycoprotein (P-gp), which facilitates the transmembrane transport of a wide range of substrates, including opioid analgesics such as morphine. This protein is expressed in several physiologically significant barrier tissues, particularly in enterocytes, hepatocytes, renal tubules, and the endothelium of the blood-brain barrier. P-glycoprotein functions as an energy-dependent efflux pump, utilizing ATP hydrolysis to actively remove substrates from the cell. It plays a key role in pharmacokinetics, influencing the processes of drug absorption, distribution, and elimination [1]. According to numerous studies, modulation of P-gp activity, whether through inhibition or induction, is one of the most important causes of drug-drug interactions, capable of significantly altering the therapeutic effect of medications [2].

P-glycoprotein, transcribed from the ABCB1 gene, is a membrane transporter that carries out the energy-dependent efflux of various xenobiotics, including morphine, from the central nervous system into the systemic circulation, thereby limiting its penetration across the blood-brain barrier. Genetic variations in ABCB1, such as the single nucleotide polymorphisms rs1045642 (C3435T), rs2032582 (G2677T/A), and rs1128503 (C1236T), are associated with altered expression levels and functional activity of P-glycoprotein. This, in turn, can significantly modify the pharmacokinetics of morphine, including its bioavailability, tissue exposure, and clinical effect [3, 4].

For example, the genetic variant rs2032582 represents a G2677T/A polymorphism in the ABCB1 gene. According to scientific publications, certain structural changes in the MDR1 gene may be associated with an increased risk of adverse reactions to morphine, such as pronounced drowsiness, confusion, and hallucinations. It has been noted that in patients carrying the guanosine (G) allele at position 2677 (exon 26), such side effects occur significantly less frequently compared to those with thymidine or adenosine alleles, which lead to other amino acid substitutions [5].

In a study conducted by Campa D et al., genotyping data from 145 Italian patients receiving morphine for pain syndrome therapy were analyzed. The authors focused on the single nucleotide polymorphisms C3435T of the ABCB1/MDR1 gene and A80G of the OPRM1 gene. A significant association (p <0.0001) was established between these genetic variants and the effectiveness of pain relief. Neither patient sex nor age, nor the administered morphine doses, showed a significant impact on the clinical effect. The main factor determining the effectiveness of analgesia was precisely the genetic characteristics, especially the ABCB1/MDR1 gene polymorphism. Patients with the T/T genotype showed a more pronounced analgesic response compared to carriers of the C/C genotype. These data confirm the relevance and importance of studying pharmacogenetic factors in individualizing pain syndrome therapy and optimizing dosages [6].

The aim of this study is to identify the association between the aforementioned ABCB1 gene polymorphisms and the plasma concentration of morphine in cancer patients receiving standard analgesic therapy.

Materials and methods

The work was carried out at the clinical site of the State Budgetary Healthcare Institution "Moscow Multidisciplinary Palliative Care Center" of the Moscow Department of Health under a scientific collaboration agreement with I.M. Sechenov First Moscow State Medical University of the Ministry of Health of Russia (FSAEI HE First MSMU named after I.M. Sechenov), concluded on October 18, 2022 (No. 515-С). The conduct of the study was approved by the local ethics committee of the University, as confirmed by the meeting protocol No. 01-21 dated January 22, 2021.

The study included 86 patients with oncological diseases (45 men and 41 women aged 30 to 78 years) who were prescribed morphine for pain syndrome control, as well as a control group of 100 individuals without oncological pathology who did not receive morphine (52 men and 48 women aged 28 to 81 years). The control group was formed at the diagnostic and treatment department of City Clinical Hospital No. 4 (Moscow). It included patients undergoing preventive medical check-ups who had no identified chronic diseases or other pathological abnormalities according to clinical, laboratory, and instrumental examinations. The "healthy" status of participants in the control group was confirmed by entries in their medical records.

Before starting therapy, the level of pain syndrome was assessed using a visual analog scale (VAS). Plasma morphine concentration was determined during the phase of achieving a stable (steady-state) drug concentration.

For genotype analysis, venous blood was collected into 4 mL tubes (size 13×75 mm) with K2EDTA, intended for hematological studies (VACUETTE®, Greiner Bio-One, Austria). To ensure uniform distribution of the anticoagulant, the tubes were inverted at least 10 times, after which the samples were frozen and stored at -20 °C until DNA extraction. DNA extraction was performed from whole blood using the "DNA-sorb-B" kit (manufactured by AmpliSens, Central Research Institute of Epidemiology, Rospotrebnadzor, Russia).

Genotyping of polymorphic variants of the ABCB1 gene — rs1128503 (C1236T), rs2032582 (G2677T), and rs1045642 (C3435T) — was carried out using real-time polymerase chain reaction with ready-made reagent kits "SNP-Screen" (manufactured by CJSC SPC "Syntol", RF). Amplification and detection were performed on the CFX96 Touch Real-Time PCR Detection System platform (Bio-Rad, USA). Determination of morphine concentration in plasma was carried out by high-performance liquid chromatography using an Agilent 1260 system (Agilent Technologies, USA), equipped with a G1312B gradient pump, G4225A degasser, G1329B autosampler, G1316A column thermostat, and an Agilent 6460 tandem mass-selective detector with a Jet Stream Electrospray ionization source.

Statistical analysis of the study data was performed using electronic computing equipment and Medcalc® software version 19.8. The normality of the distribution of quantitative variables was assessed using the Shapiro–Wilk test. In the absence of a normal distribution, nonparametric tests were applied: the Mann–Whitney test for comparing two independent samples, and the Kruskal–Wallis test for comparing three or more groups. The χ² test was used for categorical variables. The level of statistical significance was set at *p* ≤0.05.

Assessment of pain syndrome severity using the VAS scale was conducted at three time points:

• Before initiating morphine therapy (according to the epicrisis data upon admission, prior to prescribing opioid analgesics);

• Immediately before administering the next morphine dose on the day of blood sampling for the study (on days 5–7 from the start of treatment);

• At the time of reaching the maximum plasma concentration of the drug (30–90 minutes after morphine administration) [7].

Results

For a more accurate assessment of the influence of genetic variants on plasma morphine concentration, all patients were stratified into three subgroups depending on the daily dose of the drug. The first subgroup included patients receiving a fixed morphine dose of 30 mg/day; the second subgroup comprised participants receiving 40 to 60 mg/day; the third subgroup consisted of patients receiving high doses in the range of 80 to 100 mg/day.

This stratification allowed us to account for the possible dependence of pharmacokinetic parameters on dose and to minimize the influence of dose differences on the assessment of genotype impact. In all subgroups, morphine exposure indicators (median, mean value, range) were compared depending on allele and genotype carriage for the ABCB1 gene polymorphisms rs1128503, rs2032582, and rs1045642. This division made it possible to identify dose-dependent and genotype-specific differences, most pronounced in the subgroups with doses of 40–60 mg and 80–100 mg/day.

During the analysis of the possible influence of the ABCB1 gene polymorphism rs1128503 on morphine exposure, differences in the steady-state drug concentration were revealed in patients with different genotypes upon stratification by daily dose. Thus, at a dosage of 40–60 mg/day, the morphine concentration in TT genotype carriers was higher compared to heterozygotes (CT) and homozygotes for the wild-type allele (CC), with the differences between CT and TT reaching statistical significance (р ≤0.05). In the morphine subgroup of 30 mg/day, a trend towards increased concentration was observed in TT genotype carriers compared to CC and CT. In the 80–100 mg/day subgroup, no significant differences could be identified, which is associated with the absence of TT genotype carriers in this sample (Table 1).

Table 1
Steady-state morphine concentration in patients' blood depending on the ABCB1 1236 rs1128503 gene polymorphism

Analysis of genotype-specific differences for the rs2032582 polymorphism revealed significantly higher morphine concentrations in patients with the TT genotype compared to GG and GT in the 40–60 mg/day dose group (р ≤0.05). In the high-dose group (80–100 mg/day), a similar trend was also observed: in GT genotype carriers, median morphine concentrations were higher than in patients with the GG genotype. However, the differences did not reach statistical significance. In the 30 mg/day subgroup, no pronounced differences between genotypes were observed. The obtained data suggest a role for this polymorphism in the variability of morphine efflux transport (Table 2).

Table 2
Steady-state morphine concentration in patients' blood depending on the ABCB1 2677 rs2032582 gene polymorphism

The most pronounced differences in plasma morphine concentration were recorded when analyzing the rs1045642 polymorphism. In the 80–100 mg/day dose group, the drug concentration in TT genotype carriers was statistically significantly higher than in patients with CC and CT genotypes (р ≤0.05 for all pairwise comparisons). A similar pattern was observed at the 40–60 mg/day dose, although the overall χ²-test did not reach statistical significance (p=0.097). In the 30 mg/day group, no significant differences were identified. The data confirm the potential role of rs1045642 as one of the key pharmacogenetic markers influencing morphine exposure (Table 3).

Table 3
Steady-state morphine concentration in blood depending on the ABCB1 3435 rs1045642 gene polymorphism

Discussion

The obtained results confirm the role of ABCB1 polymorphisms as pharmacogenetic predictors of plasma morphine concentration. The increased exposure in TT carriers may be associated with reduced expression or activity of P-glycoprotein, which contributes to decreased clearance and enhanced systemic bioavailability of morphine. This observation has clinical significance: patients with the TT genotype are potentially more susceptible to the risk of overdose and require careful dose titration. At the same time, patients with the CC genotype may demonstrate relative resistance to therapy, necessitating dose increases or a switch to alternative opioids. The results are consistent with data from international studies and confirm observations described in the literature, demonstrating similar patterns for the first time in a Russian cohort of palliative cancer patients. Of particular importance are the identified differences for the TT genotype (rs1045642 and rs2032582), which is associated with increased morphine exposure. This may be due to reduced expression or functional activity of P-glycoprotein. This fact has direct clinical and pharmacological significance: carriers of such variants have a higher risk of accumulation and side effects, which requires an individualized approach when prescribing morphine doses. At the same time, patients with "wild-type" genotypes may exhibit relative resistance, requiring dose increases or drug replacement. Thus, the inclusion of pharmacogenetic testing in clinical practice could contribute to the personalization of opioid therapy in palliative oncology and optimize the efficacy-safety ratio.

Conclusion

This study confirms that polymorphisms in the ABCB1 gene, especially at positions rs1045642 and rs2032582, significantly contribute to interindividual differences in plasma morphine concentrations in patients with oncological diseases receiving analgesic therapy. A trend towards increased morphine exposure was identified in carriers of the TT genotype, which may be associated with reduced activity of the transporter protein P-glycoprotein encoded by ABCB1. This feature has potential for clinical application: patients with genotypes predisposing to morphine accumulation may require lower initial doses and enhanced monitoring of tolerability, whereas the presence of alleles promoting faster elimination may necessitate dose adjustments upward.

The obtained results underscore the importance of incorporating pharmacogenetic testing into the algorithm for individualizing opioid therapy, especially in palliative care settings. However, the limitations of the study must be considered, including the small sizes of individual genotypic subgroups and the need to confirm the obtained data in larger cohorts. In the future, the development of clinical recommendations for the use of pharmacogenetic testing when prescribing morphine, taking into account the ABCB1 genotype, as well as assessment of its impact not only on pharmacokinetics but also on the frequency of side effects, resistance to therapy, and patients' quality of life, appears promising.