AB0014 METHOTREXATE - IMPLICATIONS OF PHARMACOGENETICS IN THE TREATMENT OF PATIENTS WITH RHEUMATOID ARTHRITIS

Background:Methotrexate (MTX) is an anti-folate drug with anti-proliferative and anti-inflammatory effects. MTX proved to be the most highly effective, fast-acting disease modifying anti-rheumatic drug (DMARD), being widely used for the treatment of rheumatoid arthritis (RA) (1).Objectives:This review aims to describe the main genetic variants identified concerning proteins that play a role in methotrexate’s kinetics and efficiency profile.Methods:A literature review was conducted since January of 2000 until December 2020, by searching the PubMed and Embase bibliographic databases, employing the following MeSH terms: methotrexate, pharmacogenetics, pharmacokinetics and rheumatoid arthritis. The search was limited to articles in English language. Two independent reviewers screened the titles and abstracts followed by a full-text review to assess papers regarding their eligibility. A total of 48 articles matched the research criteria and were analysed.Results:Genetic variants of four main proteins, with different functions, have been consistently described.Reduced folate carrier 1 (RFC1), a constitutively expressed folate transport protein that has high affinity for MTX is responsible, almost exclusively, for the transport of folate and MTX into the cell. The most commonly studied variant of the gene is the 80G > A variant (rs1051266), mapped within exon 2, on chromosome 21. It seems to improve RA responses to MTX, clinical efficacy with long disease remission (2).ABC transporters are involved in the eflux of MTX from cells. An increased expression and function of these transporters should decrease MTX concentrations in target cells, resulting in lack of therapeutic response. ABCB1 3435 C/T (rs1045642) is a high frequency polymorphism, significantly associated with RA good responses, symptom remission and reduced adverse events, due to MTX treatment (3).Thymidylate synthase (TYMS) is involved in thymidine synthesis. MTX decreases TYMS activity by inhibition and decreasing the access to tetrahydrofolate (THF) cofactors (1). The most common genetic variant of the TYMS gene consists of a 28 bp tandem repeat (rs34743033), with double and triple number of repeats (2R and 3R). The 3R allele genotype was associated with decreased efficacy and increased toxicity (4).The 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme is indirectly inhibited by MTX. The most common SNPs of the MTHFR gene are C677T (rs1801133) and A1298C (rs1801131). Both are associated with a decreased efficacy and an increased toxicity of MTX (5).Conclusion:MTX response is affected by many gene variants; the effect of each variant separately is likely to be small. Additionally, gene-gene interaction, enhancing the potential role of linkage disequilibrium. This shows the emerging need for a better gene characterization and to improve the knowledge about variants distribution according to ethnicity, to explain different responses to MTX at an individual level.References:[1]Song, G. et al. Association of the MTHFR C677T and A1298C polymorphisms with methotrexate toxicity in rheumatoid arthritis: a meta-analysis. Clin Rheumatol33, 1715–1724 (2014).[2]Hayashi H. et al. A single nucleotide polymorphism of reduced folate carrier 1 predicts methotrexate efficacy in Japanese patients with rheumatoid arthritis. Drug Metab Pharmacokinet. 2013;28(2):164-8.[3]Zhu H. et al. Pharmacogenetics and pharmacogenomics for rheumatoid arthritis responsiveness to methotrexate treatment: the 2013 update. Pharmacogenomics. 2014 Mar;15(4):551-66.[4]Owen SA. et al. Genetic polymorphisms in key methotrexate pathway genes are associated with response to treatment in rheumatoid arthritis patients. Pharm J. 2013;13:227–34.[5]Hughes LB. et al. Racial or ethnic differences in allele frequencies of single-nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene and their influence on response to methotrexate in rheumatoid arthritis. Ann Rheum Dis. 2006;65:1213–8.Disclosure of Interests:None declared

Influence of RFC1 c.80A>G Polymorphism on Methotrexate-Mediated Toxicity and Therapeutic Efficacy in Rheumatoid Arthritis: A Meta-analysis

Background: Methotrexate (MTX) is an antirheumatic drug, transported by reduced folate carrier-1 (RFC1). The most common RFC1 gene variant, c.80 A>G (rs1051266) is ambiguously linked to adverse effects of MTX therapy in some rheumatoid arthritis (RA) patients. Objective: The purpose of meta-analysis was to summarize all major published studies on c.80 A>G SNP to clarify this ambiguity in MTX therapy. Methods: A total of 18 studies representing 3592 RA patients comprising 699 men and 2893 women were included. Both fixed and random effect models were applied to study the data. Results: The RFC1 80A-allele showed null association with MTX-mediated toxicity in both fixed (odds ratio [OR] = 0.91; 95% CI = 0.80-1.03) and random effects (OR = 0.89; 95% CI: 0.71-1.11) models. Because heterogeneity was observed in this association ( P = 0.0006), data were segregated based on use of folate therapy. In 7 studies (n = 1191) where folate was used along with MTX, RFC1 AA patients showed reduced risk for MTX-mediated toxicity (OR = 0.67; 95% CI: 0.50-0.89; P = 0.0006). The RFC1 80A-allele was found to increase the efficacy of MTX therapy by 1.53-fold (95% CI: 1.24-1.88), whereas the 80AA-genotype increased the efficacy by 1.85-fold (95% CI: 1.41-2.42). No publication bias was observed in these associations. Conclusion and Relevance: RFC1 c.80 A>G is an important pharmacogenetic determinant of MTX therapy in RA. The RFC1 80A-allele robustly increased therapeutic efficacy and safety when folate was used along with MTX. Findings are relevant to decision-making in the clinical use of MTX as a treatment for RA patients harboring the RFC1 gene variant.

Folate transporter dynamics and therapy with classic and tumor-targeted antifolates

There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.

PREVALENCE OF GLUTAMATE CARBOXYPEPTIDASE II C1561T, REDUCED FOLATE CARRIER 1 A80G, AND METHIONINE SYNTHASE A2756G GENE POLYMORPHISMS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS AMONG SOUTH INDIANS

Objective: Glutamate carboxypeptidase II (GCPII), reduced folate carrier 1 (RFC1), and methionine synthase (MTR) genes involved in the folate metabolic pathway may play a key role in the pathogenesis of diabetes and its complications. The present study aimed to investigate the prevalence of genetic polymorphisms of GCPII C1561T, RFC1 A80G, and MTR A2756G in individuals with type 2 diabetes mellitus (T2DM) among South Indians. Methods: The study subjects consisted of 100 healthy individuals and 200 patients with T2DM. Genetic polymorphisms (GCPII C1561T, RFCI A80G, and MTR A2756G) in the folate metabolic pathway were analyzed by polymerase chain reaction-restriction fragment length polymorphism method. Statistical analysis was performed to test the level of significance. Results: With regard to GCPII C1561T and MTR A2756G gene polymorphisms, significant differences were not found when diabetic patients (with and without complications) and controls were compared according to different statistical models (dominant, recessive, and overdominant) p>0.05. A case–control genetic association analysis of RFC1 A80G gene polymorphism has shown that there was 3.7-fold increased risk for patients without complications and 4.9-fold increased risk for diabetic patients with complications. Conclusions: Our findings suggest that the GCPII C1561T and MTR A2756G gene polymorphisms were not significantly associated with diabetes and its complications. Whereas, the RFCI A80G gene polymorphism involved in folate metabolism confers increased risk for diabetes and its complications in South Indian population.

Association of MTHFR and RFC1 gene polymorphisms with methotrexate efficacy and toxicity in Chinese Han patients with rheumatoid arthritis

Objective The objective was to explore the association of methylene tetrahydrofolate reductase ( MTHFR) C667T and A1298C and reduced folate carrier 1 ( RFC-1) A80G single nucleotide polymorphisms (SNP) with rheumatoid arthritis (RA) and efficacy and toxicity of methotrexate (MTX) treatment in Chinese Han patients in Henan, China. Methods Two hundred ninety-six patients with RA were enrolled (cases) and 120 healthy individuals served as controls. The genotypes of MTHFR C667T and A1298C SNP and RFC-1 A80G SNP were detected by restriction fragment length polymorphism-PCR and compared between cases and controls. We analyzed correlations of clinical effect, toxicity, and SNPs after 6 months of MTX treatment. Results We detected no significant differences in MTHFR C677T and A1298C and RFC-1 A80G SNPs between cases and controls. The RFC-1 A80G SNP differed between RA patients with good and poor efficacy after 6 months of MTX, and was an independent factor of MTX efficacy. The MTHFR C677T SNP was differently distributed in the adverse drug reaction (ADR) and non-ADR groups and was an independent factor of MTX toxicity. Conclusions In Chinese Han patients with RA, the MTHFR C667T SNP may correlate with MTX toxicity, whereas the RFC-1 A80G SNP may correlate with MTX efficacy rather than toxicity.