Pharmacogenetic markers in the treatment of patients with multidrug-resistant tuberculosis

Introduction

Despite a significant improvement in the epidemiological situation of tuberculosis (TB) in the world and the Russian Federation, the issue of treating patients with multidrug-resistant and extensively drug-resistant TB (MDR/XDR-TB) remains relevant. Treatment effectiveness rates are still far from target values: according to WHO data, for Russian patients with pre-XDR and XDR-TB in 2024, it was 54% (with a target level of 80%) [1]. This "gap" requires extensive efforts to overcome the problem, and one direction is the development of personalized patient management strategies considering individual characteristics of treatment response [2, 3].

It is assumed that the pharmacological response to treatment depends equally on phenotypic factors (sex, age, body weight, race, nature of the TB process, nutritional habits, comorbidities, immune dysfunction, drug interactions) and on genetic factors that determine the activity of enzymes and transporters involved in the biotransformation of anti-TB drugs and mediators of the immune response. Identifying these factors (polymorphisms in the corresponding genes), along with considering phenotypic features, will allow for the selection of optimal regimens and dosages of anti-TB drugs (ATDs) for the patient, improving treatment outcomes with minimal risk of toxic effects [4-7].

The composition of modern TB treatment regimens differs sharply depending on the presence of MDR in the pathogen. While for drug-susceptible TB, a number of potential pharmacogenetic markers are known [8-11], and clinical practice uses the assessment of acetylation type (genotype of N-acetyltransferase 2, a key participant in isoniazid metabolism), biomarkers for patients with MDR/XDR-TB have not been studied, and a personalized strategy has not been developed.

When searching for genetic polymorphisms — candidates for the role of a pharmacogenetic biomarker — the following conditions must be considered: 1) participation of the encoded protein in the pharmacokinetics of the drug(s); 2) association between the presence of the polymorphism and clinical effect, risk of adverse reactions (ADRs); 3) frequency in the population (at least 1%); 4) possibility of using it for dose adjustment [5].

The "core" of modern chemotherapy regimens for MDR/XDR-TB includes the so-called Group A drugs — bedaquiline, linezolid, levo- or moxifloxacin; dose adjustment is primarily justified for linezolid and fluoroquinolones. A number of enzymes and transporters are involved in the metabolism and elimination of these drugs. Key among them are the cytochrome isoforms CYP3A4 and CYP3A5, involved in the metabolism of linezolid and bedaquiline, and three main transporter proteins [12-14]: P-glycoprotein, ATP-binding cassette transporter G2, and the organic anion transporter OATP1B1 (gene SLCO1B1). For each of these proteins, encoding genes are known, mutations in which may be associated with altered pharmacological response. The possibilities of using this information in the practice of a phthisiatrician to predict and manage treatment response remain unknown.

Objective of the study: to determine pharmacogenetic markers associated with the efficacy and safety of treatment in patients with multidrug-resistant tuberculosis.

Materials and methods

This prospective cohort study included 40 patients with MDR-, pre-XDR-, and XDR-TB without HIV infection, enrolled in a treatment course including linezolid, a fluoroquinolone, and bedaquiline in 2023-2024 at the inpatient departments of the Moscow City Scientific and Practical Center for Tuberculosis Control. The cohort comprised 24 men (60.0%) and 16 women (40.0%) aged 19-66 years (median 42 years, interquartile range (IQR) 32.2-48.0 years). TB was newly diagnosed in 24 patients (60%). Among clinical forms, infiltrative TB predominated (57.5%); the proportion of patients with disseminated TB was 17.5%, with pulmonary tuberculoma — 15%, with fibrocavernous TB and caseous pneumonia — 5% each (2 patients each). Destruction cavities in the lungs were identified in 29 patients (72.5%), bacterial excretion at the start of chemotherapy — in 28 patients (70%). MDR of the pathogen was determined by microbiological and molecular genetic methods in 23 (73%) patients, pre-XDR — in 7 (17%), and XDR — in 4 patients (10%). Comorbidities were present in 35 out of 40 patients (87.5%), with pathology of the central nervous system (encephalopathy of various origins, in 17 patients, 42.5%), gastrointestinal tract (32.5%), musculoskeletal system (30%), and cardiovascular system (25%) predominating.

The chemotherapy regimen was formed according to the current version of clinical guidelines ("Tuberculosis in adults" [15]), considering the individual drug susceptibility profile of the pathogen, anamnestic data on treatment tolerability, and the spectrum and severity of concomitant pathology; all patients received bedaquiline, linezolid, and fluoroquinolones (moxifloxacin, levofloxacin, or sparfloxacin) as part of their treatment regimen, along with other drugs recommended in the regimen (cycloserine or terizidone, delamanid, prothionamide, PAS, amikacin or capreomycin, carbapenems). The spectrum of prescribed ATDs is presented in Fig. 1.

Fig. 1. Frequency of prescription of various anti-tuberculosis drugs in 40 patients with pulmonary tuberculosis (the proportion of patients in % who received each drug is indicated)

Based on literature analysis and the online knowledge base PharmGKB (https://www.pharmgkb.org/), single nucleotide polymorphisms (SNPs) in the CYP3A4, CYP3A5, ABCB1, SLCO1B1, ABCG2 genes associated with the pharmacokinetics of key ATDs for MDR-TB treatment (see Table 1), and accessible for testing by real-time polymerase chain reaction (real-time PCR), were identified.

A single 3-5 mL venous blood sample for pharmacogenetic testing was collected from all patients, regardless of treatment duration. The presence of target SNPs in transporter protein genes: SLCO1B1 (rs4149056 or T521C), ABCB1 (rs1045642 or C3435T, rs2032582 or G2677T, rs1128503 or C1236T), ABCG2 (rs2231142, C421A), as well as cytochrome CYP3A family enzymes: CYP3A4 (rs2740574, A/G), CYP3A5 (rs776746, G/A), was determined by real-time PCR using kits manufactured by NPF Syntol LLC (Russia). The follow-up period for each patient was at least 6 months (for 38 out of 40 patients, it corresponded to the duration of the intensive phase of treatment).

Treatment efficacy was assessed by the time to sputum culture conversion and the presence of positive clinical and radiological dynamics (cavity closure); safety was assessed by data on the frequency and spectrum of adverse reactions, including the presence of grade 3-4 reactions according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 (2017) [16]. The causal relationship of a reaction to a specific drug in the regimen was assessed using the Naranjo scale and expert opinion. The association of efficacy and safety indicators with the presence and variant of the studied SNPs was determined based on univariate analysis, using the χ² test, Fisher's exact test, and calculating the odds ratio (OR) and its 95% confidence interval (95% CI). Statistical data processing was performed using IBM SPSS Statistics, version 25.0.

Results

The intensive phase of treatment was successfully completed, with treatment efficacy confirmed in 37 out of 40 patients (92.5%) within 6-9 months; one of the remaining three patients died from TB progression, and two continue treatment with regimen adjustment and prolongation of the intensive phase. Cessation of bacterial excretion was noted in 25 out of 28 patients with baseline bacterial excretion (89.3%, 95% CI 72.0-97.1%) within 4 to 36 weeks from the start of chemotherapy (median 4 weeks, IQR 4-8 weeks); cavities closed in 23 out of 29 patients (79.3%, 95% CI 61.3-90.5%).

Adverse reactions were registered in 36 patients (70%, 95% CI 54.5-82.0%), of whom 62.5% (95% CI 47.0-75.8%) developed grade 3-4 ADRs requiring discontinuation of at least one ATD and modification of the treatment regimen. A total of 50 ADR cases were registered, ranging from one to six per patient. The spectrum of ADRs is shown in Fig. 2.

Fig. 2. Spectrum of adverse reactions (the percentage of patients with reactions is shown)

Neurotoxic reactions (in 13 patients, 32.5%, mainly as peripheral neuropathy) and gastrointestinal reactions (in 7 patients, 17.5%) predominated, along with arthralgia (in 12 patients, 30%). Hematological and allergic reactions (4 cases each, 10%), as well as nephro- and hepatotoxic reactions (2 patients each, 5%), occurred equally. Clinically significant QTc interval prolongation (over 500 ms) was noted in 4 patients (10%).

Results of pharmacogenetic testing (frequency of detecting various variants of the studied polymorphisms) are presented in Table 1.

Table 1. Frequency of detection of genetic polymorphisms in the study group (40 patients with tuberculosis)

It was found that the detection frequency of the studied allelic polymorphisms varied from 8.1% (for the CYP3A4 gene) to 77.8% (for the rs1128503 polymorphism in the P-glycoprotein gene). Genotypes with a mutation in both alleles of the gene (homozygous) were rare (2.7-19.4%); it was assumed that in this case, the phenotype corresponds to the most significant impairment of the encoded protein's function.

An association was found between treatment efficacy indicators and two pharmacogenetic markers: the presence of homozygous polymorphisms in the cytochrome CYP3A5 (rs776746) and ATP-binding cassette transporter G2 (rs2231142) genes.

Specifically, cessation of bacterial excretion was registered in only one of three patients with bacterial excretion carrying the AA genotype of the CYP3A5 rs776746 gene (33%) compared to 100% sputum conversion in 25 patients with the "non-mutant" genotype variant (GG), p<0.01, OR=0.021 (95% CI 0.001-0.77).

None of the patients with a homozygous mutation in the ABCG2 gene (rs2231142, genotype AA) achieved cessation of bacterial excretion within the standard timeframe of the intensive phase of chemotherapy (in one patient, bacterial excretion ceased after 9 months of treatment), while in patients with AG and GG genotypes, successful treatment outcome by the microbiological criterion was achieved in 100% of cases, p <0.01, OR=0.083 (95% CI 0.01-0.98). The AA genotype of the ABCG2 rs2231142 gene was also associated with a low rate of cavity closure: 25% vs. 75% in patients with AG and GG genotypes, p <0.01, OR=0.083, 95% CI 0.01-0.98 (see Fig. 3).

Fig. 3. Treatment efficacy rates in patients with different variants of the ABCG2 rs2231142 genotype

None of the studied polymorphisms demonstrated a statistically significant association with the overall frequency of ADRs. When analyzing possible markers for specific types of reactions, a higher frequency of neurotoxic reactions was determined in patients with the GG (wild-type) genotype of the ABCB1 rs2032582 gene (55.6% vs. 16.0% in patients with AG and AA genotypes, *p*=0.034, OR 6.3 (95% CI 1.2-33.3). A risk factor for gastrointestinal reactions was the presence of the TT genotype (homozygous mutation) in the ABCB1 rs1128503 gene: 50.0% vs. 10.0% in patients with CT and CC genotypes, *p*=0.045, OR=9.0 (95% CI 1.22-66.2%).

Furthermore, a trend towards an increased risk of hematological reactions (anemia, leukopenia, thrombocytopenia) was observed in the presence of the TT genotype of the ABCB1 rs1045642 gene (33.3% in patients with the homozygous polymorphism vs. 3.3% in patients with CC and CT genotypes); the differences were statistically non-significant — *p*=0.06 by χ² test, OR=14.5 (95% CI 1.06-198.8%). The presence of the rs4149056 polymorphism (CC or TC genotypes) of the SLCO1B1 gene, on the contrary, played a protective role: in patients with the TT genotype (absence of polymorphism, wild-type), the frequency of arthralgia was 45.5%, while in the presence of at least one "mutant" allele, no cases of arthralgia were noted (*p*=0.03 by Fisher's exact test, OR=1.79, 95% CI 1.23-2.56). Thus, polymorphisms in transporter protein genes, particularly P-glycoprotein, were of greatest significance in the genesis of ADRs.

Discussion

This study is exploratory regarding the complex of pharmacogenetic markers applicable to modern treatment regimens for patients with MDR-TB. Earlier isolated studies were devoted to studying the relationship of individual polymorphisms either with the pharmacokinetics of a specific drug [13, 14] or with clinical indicators of efficacy and safety [17, 18]. Our study did not confirm the association of treatment efficacy and ADR frequency with the CYP3A4 rs2740574 polymorphism identified in the work by Zakharov A.V. et al. [17]; at the same time, data were obtained on the potential benefit of several other biomarkers associated with the pharmacokinetics of the main drugs used in modern MDR-TB treatment regimens. In particular, the role of the CYP3A5 polymorphism identified in the work by Yunusbaeva M.M. et al. [18] was confirmed. The main result of the study is the identification of a genetic predictor of ineffective treatment — a polymorphism in the ABCG2 gene of the ATP-binding cassette transporter G. Previously, this SNP was associated only with the risk of hepatotoxicity [19]. Considering the obtained data, detection of the homozygous genotype (AA) may guide the physician towards longer treatment regimens.

In addition, allelic polymorphisms associated with the risk of significant adverse reactions (for P-glycoprotein and organic anion transporter B1 genes) were identified. Thus, a possible composition of a pharmacogenetic panel applicable for predicting treatment response and developing an optimal treatment strategy for the most complex category of patients has been determined.

This study has several limitations related to its "pilot", exploratory nature: primarily, the relatively small sample size, and secondly, the lack of analysis of phenotypic factors with potential influence on treatment outcome. Further work is planned using a larger sample size and studying the prognostic significance of polymorphisms in other enzymes and transporters associated with the efficacy and safety of treatment in patients with MDR/XDR-TB.

Conclusion

Polymorphisms in the CYP3A5 (rs776746, genotype AA) and ABCG2 (rs2231142, genotype AA) genes associated with unfavorable treatment outcomes in MDR-TB patients were identified. Genetic predictors of neurotoxic and gastrointestinal reactions during the treatment of patients with multidrug-resistant pathogen were determined.

The obtained results are applicable for identifying patients in need of individualized treatment regimens to select the most effective therapeutic strategy, as well as for early prevention of corresponding types of ADRs.