title: The Metabolism of Methazolamide in Immortalized Human Keratinocytes, HaCaT Cells.
abstract: 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 SJS/TEN.
  
  
  
  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 monooxygenase (FMO).
  
  
  
  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).
  
  
  
  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.
authors.affiliation: Department of Ophthalmology, Osaka Prefectural Medical Center for Respiratory and Allergic Diseases, 3-7-1 Habikino, Habikino-shi Osaka 583-8588. Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Sasabe
authors.affiliation: Department of Pharmacy, Osaka University Hospital, Yamadaoka, Suita, Osaka 565-0871. Japan.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Maeda
authors.affiliation: School of Comprehensive Rehabilitation, Osaka Prefecture University, Habikino, Osaka 583-8555. Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kishida
authors.affiliation: School of Comprehensive Rehabilitation, Osaka Prefecture University, Habikino, Osaka 583-8555. Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Yamano
authors.affiliation: Department of Pharmacy, Osaka University Hospital, Yamadaoka, Suita, Osaka 565-0871. Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Miwa
authors.affiliation: Department of Biochemistry, Akita University Graduate School of Medicine, Hondo, Akita 010-8543. Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Sugiyama
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000378:metabolism* / Q000627:therapeutic use / Q000633:toxicity; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D005901:Glaucoma / Q000188:drug therapy; D020719:Glucuronides / Q000378:metabolism; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D015603:Keratinocytes; D008190:Lyases / Q000378:metabolism; D008704:Methazolamide / Q000378:metabolism* / Q000627:therapeutic use / Q000633:toxicity; D008713:Methimazole / Q000494:pharmacology; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000037:antagonists & inhibitors; D013262:Stevens-Johnson Syndrome / Q000209:etiology*; D013434:Sulfenic Acids / Q000378:metabolism; D013451:Sulfonic Acids / Q000378:metabolism / Q000633:toxicity*; D053719:Tandem Mass Spectrometry / Q000379:methods; D019324:beta-Naphthoflavone / Q000494:pharmacology
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D020719:Glucuronides; D013434:Sulfenic Acids; D013451:Sulfonic Acids; D008713:Methimazole; D019324:beta-Naphthoflavone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D008190:Lyases; C019965:cystathionine beta-lyase; D003545:Cysteine; D007538:Isoniazid; D008704:Methazolamide; D000111:Acetylcysteine
keywords: 3; 4-thiadiazo-2(3H)-ylidene]acetamide; Cytochrome P450; N-[3-methyl-5-sulfo-1; Stevens-Johnson syndrome; glutathione; methazolamide; sulfenic acid; sulfonic acid; toxic epidermal necrolysis