N-demethylation of N-methyl-4-aminoantipyrine, the main metabolite of metamizole

Eur J Pharm Sci. 2018 Jul 30;120:172-180. doi: 10.1016/j.ejps.2018.05.003. Epub 2018 May 8.


Metamizole is an old analgesic used frequently in some countries. Active metabolites of metamizole are the non-enzymatically generated N-methyl-4-aminoantipyrine (4-MAA) and its demethylation product 4-aminoantipyrine (4-AA). Previous studies suggested that 4-MAA demethylation can be performed by hepatic cytochrome P450 (CYP) 3A4, but the possible contribution of other CYPs remains unclear. Using human liver microsomes (HLM), liver homogenate and HepaRG cells, we could confirm 4-MAA demethylation by CYPs. Based on CYP induction (HepaRG cells) and CYP inhibition (HLM) we could identify CYP2B6, 2C8, 2C9 and 3A4 as major contributors to 4-MAA demethylation. The 4-MAA demethylation rate by HLM was 280 pmol/mg protein/h, too low to account for in vivo 4-MAA demethylation in humans. Since peroxidases can perform N-demethylation, we investigated horseradish peroxidase and human myeloperoxidase (MPO). Horse radish peroxidase efficiently demethylated 4-MAA, depending on the hydrogen peroxide concentration. This was also true for MPO; this reaction was saturable with a Km of 22.5 μM and a maximal velocity of 14 nmol/min/mg protein. Calculation of the entire body MPO capacity revealed that the demethylation capacity by granulocyte/granulocyte precursors was approximately 600 times higher than the liver capacity and could account for 4-MAA demethylation in humans. 4-MAA demethylation could also be demonstrated in MPO-expressing granulocyte precursor cells (HL-60). In conclusion, 4-MAA can be demethylated in the liver by several CYPs, but hepatic metabolism cannot fully explain 4-MAA demethylation in humans. The current study suggests that the major part of 4-MAA is demethylated by circulating granulocytes and granulocyte precursors in bone marrow.

Keywords: Cytochrome P450 (CYP); Metamizole; Myeloperoxidase; N-demethylation; N-methyl-4-aminoantipyrine.

MeSH terms

  • Activation, Metabolic
  • Analgesics / metabolism*
  • Antipyrine / analogs & derivatives*
  • Antipyrine / metabolism
  • Cytochrome P-450 Enzyme System / metabolism
  • Demethylation
  • Dipyrone / metabolism*
  • Granulocytes / enzymology
  • HL-60 Cells
  • Hepatocytes / enzymology
  • Humans
  • Kinetics
  • Microsomes, Liver / enzymology
  • Peroxidase / metabolism
  • Substrate Specificity


  • Analgesics
  • N-methyl-4-aminoantipyrine
  • Dipyrone
  • Cytochrome P-450 Enzyme System
  • Peroxidase
  • Antipyrine