Objective: Drug therapy is occasionally accompanied by an idiosyncratic severe toxicity,
which occurs very rarely, but can lead to patient mortality. Methazolamide, an anti-glaucomatous agent,
could cause severe skin eruptions called Stevens-Johnson syndrome/toxic epidermal necrolyis
(SJS/TEN). Its precise etiology is still uncertain. In this study, the metabolism of methazolamide was
investigated in immortalized human keratinocytes to reveal the possible mechanism which causes
Methods: The metabolism of methazolamide was studied using immortalized human keratinocytes,
HaCaT cells. HPLC was used to isolate a metabolite from the culture medium. Mass spectrometry (LCMS/
MS) was employed for its characterization. Three typical chemical inducers were assessed for the
inducibility of cytochrome P450, and methimazole was used as the inhibitor of flavin-containing
Results: A sulfonic acid, N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) was
identified as the final metabolite. Dexamethasone and β-naphthoflavone behaved as an inducer of cytochrome
P450 in the metabolism, but isoniazid did not. The effect of methimazole was not consistent.
We did not detect any glucuronide nor any mercapturic acid (N-acetylcysteine conjugate).
Conclusion: N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) is not considered to
be a direct product of an enzymatic reaction, but rather an auto-oxidation product of N-[3-methyl-5-
sulfe-1,3,4-thiadiazol-2(3H)-ylidene]acetamide, a chemically unstable sulfenic acid, which is produced
by cytochrome P450 from the β-lyase product of cysteine conjugate of methazolamide. MSO is considered
to be susceptible to glutathione and to return to glutathione conjugate of methazolamide, forming a
futile cycle. A hypothetical scenario is presented as to the onset of the disease.