Metabolism studies of a small-molecule tumor necrosis factor-alpha (TNF-α) inhibitor, UTL-5b (GBL-5b)

  • Jiajiu Shaw
  • Brian Shay
  • Jack Jiang
  • Frederick Valeriote
  • Ben Chen
Original Paper


UTL-5b is an anti-inflammatory and anti-arthritic small-molecule tumor necrosis factor-alpha inhibitor and a structural analogue of the anti-arthritic drug, leflunomide. Leflunomide is known to be metabolized to teriflunomide, but the metabolites of UTL-5b have not been reported. The objective of this study was to investigate whether UTL-5b has a similar metabolic behavior as leflunomide. Preliminary studies showed that when exposed to microsomes in vitro with or without NADPH, UTL-5b disappeared within 30 min. To further investigate the microsomal metabolism, liquid chromatography-ultraviolet (LC-UV) and LC/tandem mass spectrometry (LC–MS/MS) were employed to, respectively, monitor the microsomal metabolites and identify the structure of the metabolites using LC-full scan MS and LC combined with multiple-ion monitoring MS. Fragmentation determination was analyzed by two types of scans: product ion scans and precursor ion scan. The in vitro microsomal treatment of UTL-5b resulted in two major metabolites: 5-methylisoxazole-3-carboxylic acid and 2-chloroaniline. Thus, the in vitro metabolic behavior of UTL-5b appears to be different from that of leflunomide in that the isoxazole ring is cleaved.


Microsome Metabolites LC–MS/MS TNF-α inhibitor UTL-5b 



Dihydroorotate dehydrogenase


Electrospray ionization


High performance liquid chromatography


Liquid chromatography


Liquid chromatography/tandem mass spectrometry


Multiple ion monitoring


Mass spectrometry/mass spectrometer


Nuclear magnetic resonance


Retention time


Single ion recording


Total ion current


Tumor necrosis factor-alpha





This work was supported by SBIR grant 1R43 AR052193 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Heath and Human Services, USA.


  1. Ansede JH, Voyksner RD, Ismail MA, Boykin DW, Tidwell RR, Hall JE (2005) In vitro metabolism of an orally active O-methyl amidoxime prodrug for the treatment of CNS trypanosomiasis. Xenobiotica 35(3):211–226. doi: 10.1080/00498250500087671 PubMedCrossRefGoogle Scholar
  2. Brent RL (2001) Teratogen update: reproductive risks of leflunomide (Arava); a pyrimidine synthesis inhibitor: counseling women taking leflunomide before or during pregnancy and men taking leflunomide who are contemplating fathering a child. Teratology 63(2):106–112. doi: 10.1002/1096-9926(200102)63:2<106:AID-TERA1017>3.0.CO;2-R PubMedCrossRefGoogle Scholar
  3. Bruneau JM, Yea CM, Spinella-Jaegle S, Fudali C, Woodward K, Robson PA, Sautes C, Westwood R, Kuo EA, Williamson RA, Ruuth E (1998) Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide. Biochem J 336(Pt 2):299–303PubMedGoogle Scholar
  4. Goldenberg MM (1999) Leflunomide, a novel immunomodulator for the treatment of active rheumatoid arthritis. Clin Ther 21(11):1837–1852. doi: 10.1016/S0149-2918(00)86732-6 (discussion 1821)PubMedCrossRefGoogle Scholar
  5. Hejtmancik MR, Trela BA, Kurtz PJ, Persing RL, Ryan MJ, Yarrington JT, Chhabra RS (2002) Comparative gavage subchronic toxicity studies of o-chloroaniline and m-chloroaniline in F344 rats and B6C3F1 mice. Toxicol Sci 69(1):234–243PubMedCrossRefGoogle Scholar
  6. Huang WH, Yang CL, Lee AR, Chiu HF (2003) Leflunomide analogues as potential antiinflammatory agents. Chem Pharm Bull (Tokyo) 51(3):313–314. doi: 10.1248/cpb.51.313 CrossRefGoogle Scholar
  7. Kalgutkar AS, Nguyen HT, Vaz AD, Doan A, Dalvie DK, McLeod DG, Murray JC (2003) In vitro metabolism studies on the isoxazole ring scission in the anti-inflammatory agent lefluonomide to its active alpha-cyanoenol metabolite A771726: mechanistic similarities with the cytochrome P450-catalyzed dehydration of aldoximes. Drug Metab Dispos 31(10):1240–1250. doi: 10.1124/dmd.31.10.1240 PubMedCrossRefGoogle Scholar
  8. Magne D, Mezin F, Palmer G, Guerne PA (2006) The active metabolite of leflunomide, A77 1726, increases proliferation of human synovial fibroblasts in presence of IL-1beta and TNF-alpha. Inflamm Res 55(11):469–475. doi: 10.1007/s00011-006-5196-x PubMedCrossRefGoogle Scholar
  9. Osiri M, Shea B, Robinson V, Suarez-Almazor M, Strand V, Tugwell P, Wells G (2003) Leflunomide for the treatment of rheumatoid arthritis: a systematic review and metaanalysis. J Rheumatol 30(6):1182–1190. doi: 0315162X-30-1182[pii] PubMedGoogle Scholar
  10. Shaw J, Liu C, Martin R, Chen B, Holtz R, Huang WH, Lee AR (2007) Inhibition of tumour necrosis factor-alpha secretion from EpiDermTM tissues by a novel small molecule, UTL-5d. Br J Dermatol 157(3):575–579. doi: 10.1111/j.1365-2133.2007.08025.x PubMedCrossRefGoogle Scholar
  11. Shaw J, Chen B, Huang W-H, Lee A-R, Media J, Valeriote F (2011a) The small-molecule TNF-α modulator, UTL-5g, reduces side effects induced by cisplatin and enhances the therapeutic effects of cisplatin in vivo. J Exp Ther Oncol 9(2):129–137PubMedGoogle Scholar
  12. Shaw J, Chen B, Wooley P, Huang WH, Lee AR, Zeng D (2011b) Anti-inflammatory and anti-arthritic effects of a novel leflunomide analogue, UTL-5b (GBL-5b). Am J Biomed Sci 3(1):31–39. doi: 10.5099/aj110100031 PubMedCrossRefGoogle Scholar
  13. Shaw J, Chen B, Wooley P, Palfey B, Lee AR, Huang WH, Zeng D (2011c) A novel leflunomide analog, UTL-5b (GBL-5b), suppresses JAK3, MAP3K2, and LITAF genes. Am J Biomed Sci 3(3):218–227. doi: 10.5099/aj110300219 CrossRefGoogle Scholar
  14. Strand V, Cohen S, Schiff M, Weaver A, Fleischmann R, Cannon G, Fox R, Moreland L, Olsen N, Furst D, Caldwell J, Kaine J, Sharp J, Hurley F, Loew-Friedrich I (1999) Treatment of active rheumatoid arthritis with leflunomide compared with placebo and methotrexate. Leflunomide Rheumatoid Arthritis Investigators Group. Arch Intern Med 159(21):2542–2550PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag France 2011

Authors and Affiliations

  • Jiajiu Shaw
    • 1
    • 6
  • Brian Shay
    • 2
  • Jack Jiang
    • 3
  • Frederick Valeriote
    • 4
  • Ben Chen
    • 5
  1. 1.Unitech Pharmaceuticals, Inc.Ann ArborUSA
  2. 2.Department of ChemistryWayne State UniversityDetroitUSA
  3. 3.GeneBlue CorporationSan GabrielUSA
  4. 4.Department of Internal MedicineHenry Ford Health SystemDetroitUSA
  5. 5.Department of Internal MedicineWayne State UniversityDetroitUSA
  6. 6.21st Century Therapeutic, Inc.FerndaleUSA

Personalised recommendations