Hider, S. L., Bruce, I. N. & Thomson, W. The pharmacogenetics of methotrexate. Rheumatology 46, 859 (2007).

Slørdal, L., Kolmannskog, S., Prytz, P. S., Moe, P. J. & Aarbakke, J. Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate after high-dose (33.6 g/m2) methotrexate therapy. Pediatr. Hematol. Oncol. 3, 127–134 (1986).

Article 
PubMed 

Google Scholar
 

Maryam, H., Meenal, M. Methotrexate. National Library of Medicine (2024). https://www.ncbi.nlm.nih.gov/books/NBK556114/.

Nagamine, A. et al. Target concentration achievement for efficacy and safety of patients with osteosarcoma treated with high-dose methotrexate based on individual pharmacokinetics: a retrospective study. Oncol. Lett. 25, 70 (2022).

Article 
PubMed 
PubMed Central 

Google Scholar
 

Ramsey, L. B. et al. Consensus guideline for use of glucarpidase in patients with high-dose methotrexate induced acute kidney injury and delayed methotrexate clearance. Oncologist 23, 52–61 (2018).

Article 
CAS 
PubMed 

Google Scholar
 

Roth, M., Obaidat, A. & Hagenbuch, B. OATPs OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. Br. J. Pharmacol. 165, 1260–1287 (2012).

Choughule, K. V., Joswig-Jones, C. A. & Jones, J. P. Interspecies differences in the metabolism of methotrexate: an insight into the active site differences between human and rabbit aldehyde oxidase. Biochem. Pharmacol. 96, 288–295 (2015).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Dervieux, T. et al. HPLC determination of thiopurine nucleosides and nucleotides in vivo in lymphoblasts following mercaptopurine therapy. Clin. Chem. 48, 61–68 (2002).

Article 
CAS 
PubMed 

Google Scholar
 

Grim, J., Chladek, J. & Martinkovi, J. Pharmacokinetics and pharmacodynamics of methotrexate in non-neoplastic diseases. Clin. Pharmacokinet. 42, 139–151 (2003).

Article 
CAS 
PubMed 

Google Scholar
 

Prasad, B. et al. Ontogeny of hepatic drug transporters as quantified by LC-MS/MS Proteomics. Clin. Pharmacol. Ther. 100, 362–370 (2016).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Dette, C. M. U. et al. Advancing rare disease therapeutics through digital twins: opportunities in drug development and precision dosing. Comput. Struct. Biotechnol. J. 28, 592–608. https://doi.org/10.1016/j.csbj.2025.11.047 (2025).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Britz, H. et al. Physiologically based pharmacokinetic models of probenecid and furosemide to predict transporter mediated drug-drug interactions. Pharm. Res. 37, 250 (2020).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

European Medicines Agency: EMA/CHMP/458101/2016 – Reporting of physiologically based pharmacokinetic (PBPK) modelling and simulation (2019). https://www.ema.europa.eu/en/reporting-physiologically-based-pharmacokinetic-pbpk-modelling-simulation-scientific-guideline.

U.S. Food and Drug Administration: FDA-2016-D-3969 – Physiologically Based Pharmacokinetic Analyses — Format and Content Guidance for Industry (2018). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/physiologically-based-pharmacokinetic-analyses-format-and-content-guidance-industry.

Wojtyniak, J., Britz, H., Selzer, D., Schwab, M. & Lehr, T. Data digitizing: accurate and precise data extraction for quantitative systems pharmacology and physiologically‐based pharmacokinetic modeling. CPT Pharmacometr. Syst. Pharmacol. 9, 322–331. https://doi.org/10.1002/psp4.12511 (2020).

Article 
CAS 

Google Scholar
 

Chemicalize Methotrexate Entry. https://chemicalize.com/app/calculation (2026).

Chen et al. Pubchem 2025 update. Nucleic Acids Res. (2025). https://pubchem.ncbi.nlm.nih.gov/compound/5484402#section=Chemical-and-Physical-Properties.

Chemicalize 7-Hydroxymethotrexate Entry. https://chemicalize.com/app/calculation (2026).

Uhlén, M. et al. Tissue-based map of the human proteome. Science (1979) 347, 8596 (2015).

Fisher, R. S., Rock, E. & Malmud, L. S. Effects of meal composition on gallbladder and gastric emptying in man. Dig. Dis. Sci. 32, 1337–1344 (1987).

Article 
CAS 
PubMed 

Google Scholar
 

Selzer, D., Rüdesheim, S., Dette, C. M. U., Marok, F. Z. & Lehr, T. Ospgsa: global sensitivity analysis for open systems pharmacology models (2026). https://github.com/Clinical-Pharmacy-Saarland-University/ospgsa.

Borgonovo, E. A new uncertainty importance measure. Reliab. Eng. Syst. Saf. 92, 771–784 (2007).

Article 

Google Scholar
 

De Carlo, A., Tosca, E. M., Melillo, N. & Magni, P. A two-stages global sensitivity analysis by using the δ sensitivity index in presence of correlated inputs: application on a tumor growth inhibition model based on the dynamic energy budget theory. J. Pharmacokinet. Pharmacodyn. 50, 395–409 (2023).

Article 
PubMed 
PubMed Central 

Google Scholar
 

Cuquerella-Gilabert, M. et al. Leveraging two‐stage δ global sensitivity analysis method to inform parameter estimation in PBPK models. Pharm Stat 25, 859 (2026).

Jamaladdin, N. et al. Key pharmacokinetic parameters of 74 pediatric anticancer drugs providing assistance in preclinical studies. Clin. Pharmacol. Ther. 114, 904–913 (2023).

Article 
CAS 
PubMed 

Google Scholar
 

Dong, W. C. et al. Simultaneous determination of unbound plasma concentration of methotrexate and 7-hydroxymethotrexate in children patients receiving high-dose methotrexate therapy. Drug Des. Devel Ther. 19, 4383–4396. https://doi.org/10.2147/DDDT.S516431 (2025).

Article 
PubMed 
PubMed Central 

Google Scholar
 

Hunt, J. P. et al. Maximum likelihood estimation of renal transporter ontogeny profiles for pediatric PBPK modeling. CPT Pharmacometr. Syst. Pharmacol. 13, 576–588 (2024).

Article 
CAS 

Google Scholar
 

Open Systems Pharmacology Suite Community. PK-Sim® Ontogeny Database Documentation, Version 7.3 (2024). https://github.com/Open-Systems-Pharmacology/OSPSuite.Documentation/blob/master/PK-Sim%20Ontogeny%20Database%20Version%207.3.pdf.

Namour, F. et al. Pharmacokinetics, safety, and tolerability of GLPG0259, a Mitogen-Activated Protein Kinase-Activated Protein Kinase 5 (MAPKAPK5) inhibitor, given as single and multiple doses to healthy male subjects. Drugs R D. 12, 141–163. https://doi.org/10.2165/11633120-000000000-00000 (2012).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Stoller, G. R., Samuel, A. J., James, C. D., Robert, J. L. & Bruce, A. C. Pharmacokinetics of high-dose methotrexate (NSC-740). Rep. Div. Cancer Treat. NCI. 2, 285–290 (1976).


Google Scholar
 

U.S. Food and Drug Administration: FDA-2010-D-0133 – Pharmacokinetics in Patients with Impaired Renal Function — Study Design, Data Analysis, and Impact on Dosing 8 (2024). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/pharmacokinetics-patients-impaired-renal-function-study-design-data-analysis-and-impact-dosing.

Guest, E. J., Aarons, L., Houston, J. B., Rostami-Hodjegan, A. & Galetin, A. Critique of the two-fold measure of prediction success for ratios: application for the assessment of drug-drug interactions. Drug Metab. Dispos. 39, 170–173 (2011).

Article 
CAS 
PubMed 

Google Scholar
 

Rajagopalan, P. T. R. et al. Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics. Proc. Natl. Acad. Sci. U S A. 99, 13481 (2002).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Jones, H. & Rowland-Yeo, K. Basic concepts in physiologically based pharmacokinetic modeling in drug discovery and development. CPT Pharmacometrics Syst. Pharmacol. 2, 1–12 (2013).

Article 

Google Scholar
 

Schaller, S. et al. Evaluation of BCRP-related DDIs between methotrexate and cyclosporin A using physiologically based pharmacokinetic modelling. Drugs R D. 25, 1–17. https://doi.org/10.1007/s40268-024-00495-1 (2024).

Article 
PubMed 
PubMed Central 

Google Scholar
 

Hwang, S. et al. Comprehensive evaluation of OATP- and BCRP‐mediated drug–drug interactions of methotrexate using physiologically‐based pharmacokinetic modeling. Clin. Pharmacol. Ther. 116, 1013–1022 (2024).

Article 
CAS 
PubMed 

Google Scholar
 

Aherne, G. W., Piall, E., Marks, V., Mould, G. & White, W. F. Prolongation and enhancement of serum methotrexate concentrations by probenecid. BMJ 1, 1097–1099 (1978).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Tracy, T. S. et al. The effects of a salicylate, ibuprofen, and naproxen on the disposition of methotrexate in patients with rheumatoid arthritis. Eur. J. Clin. Pharmacol. 42, 121–125 (1992).

Article 
CAS 
PubMed 

Google Scholar
 

Servier Medical Art by Servier is licensed under a Creative Commons Attribution 4.0 Unported License. https://creativecommons.org/licenses/by/4.0/ (2026).

Skibińska, L., Ramlau, C., Załuski, J. & Olejniczak, B. Methotrexate binding to human plasma proteins. Pol. J. Pharmacol. Pharm. 42, 151–157 (1990).

PubMed 

Google Scholar
 

Slørdal, L., Sager, G., Jæger, R. & Aarbakke, J. Interactions with the protein binding of 7-hydroxy-methotrexate in human serum in vitro. Biochem. Pharmacol. 37, 607–611 (1988).

Article 
PubMed 

Google Scholar
 

Yousefi, G., Foroutan, S. M., Zarghi, A. & Shafaati, A. Synthesis and characterization of methotrexate polyethylene glycol esters as a drug delivery system. Chem. Pharm. Bull. (Tokyo). 58, 147–153 (2010).

Article 
CAS 
PubMed 

Google Scholar
 

Mir, O. et al. Hyper-alkalinization without hyper-hydration for the prevention of high-dose methotrexate acute nephrotoxicity in patients with osteosarcoma. Cancer Chemother. Pharmacol. 66, 1059–1063 (2010).

Article 
CAS 
PubMed 

Google Scholar
 

Chen, Z. S. et al. Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport. Cancer Res. 63, 4048–4054 (2003).

CAS 
PubMed 

Google Scholar
 

Inoue, K. & Yuasa, H. Molecular basis for pharmacokinetics and pharmacodynamics of methotrexate in rheumatoid arthritis therapy. Drug Metab. Pharmacokinet. 29, 12–19 (2014).

Article 
CAS 
PubMed 

Google Scholar
 

Patik, I. et al. Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate. Biochem. Pharmacol. 98, 649–658 (2015).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Ifergan, I., Jansen, G. & Assaraf, Y. G. The Reduced Folate Carrier (RFC) is cytotoxic to cells under conditions of severe folate deprivation. J. Biol. Chem. 283, 20687–20695. https://doi.org/10.1074/jbc.M802812200 (2008).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Bedoui, Y. et al. Methotrexate an old drug with new tricks. Int. J. Mol. Sci. 20, 5023. https://doi.org/10.3390/ijms20205023 (2019).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Tanaka, G. & Kawamura, H. Anatomical and physiological characteristics for Asian reference man: Male and female of different ages: Tanaka model. Report Number: NIRS-M-115. In Division of Radioecology, National Institute of Radiological Sciences. Hitachinaka 311 – 12 Japan. (1996).

Valentin, J. Basic anatomical and physiological data for use in radiological protection: reference values. Ann. ICRP. 32, 1–277 (2002).

Article 

Google Scholar
 

National Center for Health Statistics. Third National Health and Nutrition Examination Survey, (NHANES III) (1997). https://wwwn.cdc.gov/nchs/nhanes/nhanes3/default.aspx.

Ishii, E. et al. Oral mucositis and salivary methotrexate concentration in intermediate-dose methotrexate therapy for children with acute lymphoblastic leukemia. Med. Pediatr. Oncol. 17, 429–432 (1989).

Article 
CAS 
PubMed 

Google Scholar
 

Ylinen, E., Jahnukainen, K., Saarinen-Pihkala, U. M. & Jahnukainen, T. Assessment of renal function during high-dose methotrexate treatment in children with acute lymphoblastic leukemia. Pediatr. Blood Cancer. 61, 2199–2202. https://doi.org/10.1002/pbc.25137 (2014).

Article 
CAS 
PubMed 

Google Scholar
 

Winograd, B. et al. Renal excretion and pharmacokinetics of methotrexate and 7-hydroxy-methotrexate following a 24-h high dose infusion of methotrexate in children. Eur. J. Clin. Pharmacol. 30, 231–238 (1986).

Article 
CAS 
PubMed 

Google Scholar
 

Kearney, P. J., Light, P. A., Preece, A. & Mott, M. G. Unpredictable serum levels after oral methotrexate in children with acute lymphoblastic leukaemia. Cancer Chemother. Pharmacol. 3, 859 (1979).

Peters, S. A. & Dolgos, H. Requirements to Establishing Confidence in Physiologically Based Pharmacokinetic (PBPK) models and overcoming some of the challenges to meeting them. Clin. Pharmacokinet. 58, 1355–1371 (2019).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Taylor, Z. L. et al. Systematic review of pharmacogenetic factors that influence high-dose methotrexate pharmacokinetics in pediatric malignancies. Cancers (Basel). 13, 2837. https://doi.org/10.3390/cancers13112837 (2021).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Hutzler, J. M., Yang, Y. S., Brown, C., Heyward, S. & Moeller, T. Aldehyde oxidase activity in donor-matched fresh and cryopreserved human hepatocytes and assessment of variability in 75 donors. Drug Metab. Dispos. 42, 1090–1097 (2014).

Article 
PubMed 

Google Scholar
 

Foti, A., Dorendorf, F. & Leimkühler, S. A single nucleotide polymorphism causes enhanced radical oxygen species production by human aldehyde oxidase. PLoS One. 12, e0182061. https://doi.org/10.1371/journal.pone.0182061 (2017).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Ogungbenro, K. & Aarons, L. Physiologically based pharmacokinetic modelling of methotrexate and 6-mercaptopurine in adults and children. Part 1: methotrexate The CRESim & Epi-CRESim Project Groups. J. Pharmacokinet. Pharmacodyn. 41, 159–171. https://doi.org/10.1007/s10928-014-9354-4 (2026).

Le Merdy, M., Mullin, J. & Lukacova, V. Development of PBPK model for intra-articular injection in human: methotrexate solution and rheumatoid arthritis case study. J. Pharmacokinet. Pharmacodyn. 48, 909–922 (2021).

Article 
PubMed 
PubMed Central 

Google Scholar
 

Brandhonneur, N., Noury, F., Bruyère, A., Saint-Jalmes, H. & Le Corre, P. PBPK model of methotrexate in cerebrospinal fluid ventricles using a combined microdialysis and MRI acquisition. Eur. J. Pharm. Biopharm. 104, 117–130 (2016).

Article 
CAS 
PubMed 

Google Scholar
 

Batey, M. A. et al. Population pharmacokinetics of adjuvant cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Eur. J. Cancer. 38, 1081–1089 (2002).

Article 
CAS 
PubMed 

Google Scholar
 

Campagne, O. et al. Population pharmacokinetics of methotrexate and 7-hydroxymethotrexate and delayed excretion in infants and young children with brain tumors. Eur. J. Pharm. Sci. 193, 106669 (2024).

Article 
CAS 
PubMed 

Google Scholar
 

Fukuhara, K., Ikawa, K., Morikawa, N. & Kumagai, K. Population pharmacokinetics of high-dose methotrexate in Japanese adult patients with malignancies: a concurrent analysis of the serum and urine concentration data. J. Clin. Pharm. Ther. 33, 677–684 (2008).

Article 
CAS 
PubMed 

Google Scholar
 

Korell, J. et al. A Population pharmacokinetic model for low-dose methotrexate and its polyglutamated metabolites in red blood cells. Clin. Pharmacokinet. 52, 475–485 (2013).

Article 
CAS 
PubMed 

Google Scholar
 

Pesenti, G., Foppoli, M. & Manca, D. A minimal physiologically based pharmacokinetic model for high-dose methotrexate. Cancer Chemother. Pharmacol. 88, 595–606. https://doi.org/10.1007/s00280-021-04305-2 (2021).

Article 
CAS 
PubMed 
PubMed Central 

Google Scholar
 

Mould, D. & Upton, R. Basic concepts in population modeling, simulation, and model-based drug development—part 2: introduction to pharmacokinetic modeling methods. CPT Pharmacometrics Syst. Pharmacol. 2, 1–14 (2013).

Article 

Google Scholar
 

Darwich, A. S. Model-informed precision dosing: background, requirements, validation, implementation, and forward trajectory of individualizing drug therapy. Annu. Rev. Pharmacol. Toxicol. 61, 225–245 (2021).

Article 
CAS 
PubMed 

Google Scholar