The Metabolic Fate of Tolbutamide in Man and in the Rat - Journal of

John E. Toth, Gerald B. Grindey, William J. Ehlhardt, James E. Ray, George B. Boder, Jesse R. Bewley, Kim K. Klingerman, Susan B. Gates, Sharon M. Rin...
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~IETABOLISU OF TOLBUTAMIDE

July 1966

507

The Metabolic Fate of Tolbutamide in Man and in the Rat RICHARD C. THOMAS AND GEORGE J. IKEDA Biochemical Research Division, The Upjohn Company, K a l a m z o o , Michigan Received January 28, 1966 Tritium-labeled tolbutamide was found to be metabolized b y man to l-butyl-3-(p-hydroxymethyl)phenylsulfonylurea as well as to the generally recognized metabolite, 1-butyl 3-(p-carboxy)phenylsnlfonylurea. These two metabolites NTere the only drug-related materials detected in urine and together comprised 85% of an orally administered dose of tolbutamide. Both metabolites were isolated from iirine in crystalline form and characterized. The carboxy metabolite accounted for 677, and the hydroxymethyl metabolite 337, of urinary radioactivity as determined by quantitative paper and thin layer chromatography. I n the rat, 80% or" an orally administered dose of tritium-labeled tolbutamide was excreted in urine, predominantly as l-butyl-3-( p-hydroxymethy1)phenylsulfonylurea, which was isolated in crystalline form and characterized. Small amounts of l-butyl-3-(p-carboxy)phenylsulfonylurea and p-tolylsulfonylurea (together approximately 5 7 , of drug-related material excreted in urine) were also detected by paper chromatography.

The metabolism of tolbutamide (1-butyl-3-p-tolylsulfonylurea, I), an orally active hypoglycemic agent,

-

H , C ~ S ONHCO , NH (CHMH,

I

has been extensively studied.' The literature, although occasionally in conflict, indicates pronounced species differences. The major metabolite of tolbutamide in man has been isolated and identified as l-butyl-3-pcarboxyphenylsu1fonylurealaLc (carboxy metabolite, 11),

1 U

HOOC

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SO,NHCONH(CHz),CH,

I1

although Wittenhagen, et aZ.,ld on the basis of paper chromatography, reported that 1-butyl-3-p-hydroxymethylphenylsulfonylurea (hydroxymethyl metabolite, 111) is also produced in small amounts. The dog is

-

HOCH,~,NHCONH(CH~),CH,

I11

reportedldVe to excrete p-tolylsulfonamide and p-tolylsulfonylurea following oral administration of tolbutamide. Larsen and IIadsenl' mention I11 as the primary metabolite of tolbutamide in the cat, but do not elaborate on its identification. Wittenhagen, et aZ.,ld however, state that preliminary paper chromatographic results indicated that the cat metabolizes tolbutamide in a manner similar to the dog. Miller, et aZ.,lgemploying paper chromatography, found that the rat converts (1) L. H . Louis. S. S. Fajans, J. W.Cpnn, W. A . Struck, J. B. Wright, a n d J. L. Johnson, J . Am. Chem. Soc., 78, 5701 (1956); (b) G . Wittenhagen and G. Mohnike, Deut. Med. Wochschr., 81, 887 (1956); ( c ) T . Dorfmiiller, ibid 82, 888 (1956); (d) G. Wittenhagen, G . Mohnike, and W.Langenbeck, Z . Phusiol. Chem., 816, 157 (1959); (e) G. Mohnike, G. Wittenhagen, and JV. Langenbeck, Naturwiss., 45, 13 (1958); (f) J. A. Larsen and J. Madsen. Proc. SOC.E z p t l . B i d . M e d . , 109, 120 (1962); ( 9 ) W. L. Miller, Jr., J. J. Krake, M. J. VanderBrook, a n d L. M. Reineke, Ann. N . Y . Acad. Sci., 71, 118 (1957); (h) S. S. Fajans, L. H . Louis, H . S. Seltzer, R. D. Johnson, R. D . Gittler, A. R. Hennes, B. L. Wajchenberg, I. P. Ackerman, a n d J. W. Conn, Metab. Clin. Ezptl.. 6, 820 (1956); (i) G. Mohnike and G. Wittenhagen, Deut. Med. Wochschr., 82, 1556 (1957); (j) R. U. Lemieux, K. F. Sporek, I. O'Reilly, and E. Nelson, Biochem. Pharmacol., 7, 31 (1961); (k) E. Nelson a n d I. O'Reilly, J . Pharmacol. E z p t l . Therap., 182, 103 (1961); (1) E. Nelson, Sature, 195, 76 (1962); (m) E. Nelson. S. Long, a n d J. G. Wagner, J . Pharm. Sci., 53, 1224 (1962); (n) B. E. Ballard and E. Nelson, Arch. Intern. Pharmacodyn., 133, 206 (1961).

S35-labeled tolbutamide to a major and two minor metabolites. One of the minor metabolites appeared to be 11, but the others, including the major metabolite, in the light were not identified. Wittenhagen, et of their own work, stated that the unidentified excretion products are I11 and unchanged tolbutamide. Inspection of Miller's results, however, shons that no unchanged tolbutamide is excreted by the rat. Wittenhagen, et al.,ld also report that tolbutamide is converted to I1 by rats, guinea pigs, and rabbits. In each case a minor amount of I11 was detected in the animal's urine. I n each case that has implicated I11 as a metabolite of tolbutamide, identification has been by paper chromatography in a single solvent system; crystalline material has not been isolated and subjected to analysis. Although I11 has been reported as a metabolite of tolbutamide in man, there has been no investigation of its quantitative importance relative to 11. Previous efforts to quantify I1 in urine have involved either its or use''-" of an direct precipitation from assay2 which, as will be discussed, also responds to 111. The present work, employing tritium-labeled tolbutamide, was conducted to clarify both qualitatively and quantitatively the metabolism of this drug in man and in the rat.

Experimental Section Radioactivity Measurements.-All counting was performed with a Tri-Cub3 Model 314X or 314EX-2A liquid scintillation spectrometer a t - 8" under conditions suitable for measuring tritium. Appropriate aliquots of samples were dissolved in 15 ml of Diotol scintillation solvent [toluene-dioxane-methanol (350:350:210 by vol) containing 73 g of naphthalene, 4.6 g of 2,5diphenyloxazole, and 0.080 g of 1,4-bis-2-(5-phenyloxazoly1)benzene/l.]. The absolute counting efficiency for each sample was determined by addition of an internal standard of tritiumlabeled toluene and results then were converted to microcuries or disintegrations per minute. Paper and Thin Layer Chromatography.-Paper chromatography was carried out in a 1-butanol-piperidine-water (81: 2 : 17 by vol) system on Whatman No. 1 paper. Dried chromatograms were routinely examined under short wavelength ultraviolet light with an ultraviolet scanner4 to locate standards and metabolites, when possible, by fluorescence quenching. Radioactive zones were located by cutting the developed paper (2) E. Nelson, I. O'Reilly, and T. Chulski, Clin. Chim. Acta, 6, 7 i 4 (1960). (3) Packard Instrument Co., Inc., Downers Grove, Ill. (4) N. A. Drake, W.J. Haines, R . E. Knauff, and E. D. Nielson, Anal. Chem., 28, 2036 (1956).

strip into sequeiitial 1.25- or 2.5-cm segnieiits (deperiding on the resoliitioii desired) and coutitiiig the segmeiits iii iiidivit1ii:il i~oiiiitiiigvials in the iisiial mariiier. Thiii layer chromatogra . w : i q c:trrietl o i i t iii it vhloroforni,ten1 0 1 ' iti :i i,liliiriifiiri~~--nietliaiiiil--flJ~lIlicncid (92:s hy vol) fomiic acid ('35:4: 1 I J ~vol) system 011 silica gel GF.6 The fliioresceiice queiichiiig of staiidartls aiid, when pii41)le) met at,olitea was detected bj- viewing the dried c,hronintogrnrris iiiider short-wavelength iiltraviolet light. Itndioactive zi)iies \wre 1111:atedI)y traiisferrnig seyueiitial 0.62- 111' 1.25-ciii aegmeiit > (depending on the resolution desired) of the developed chrornatogram irito iiidividiial coiiriting vials :mil ci~iiiitiiigiii the i i 5 i i : i I manner. Rat Experiments.--Tolbutariiide wa.~,u~petided iii water :iiid iheri dissolved by additioii of a .light e s c e i s of XHIOH. T h r pM of the solutioii ~ : t hadjuqted to :ipprosiiii:itely 7 . 5 :iiid theii dilii(etl so ah t i l c*oiit:iiii GS rng of toll~ur:~niide/tiil. Trit iuriilalvlcd ti~lI~iii:i~iiide \?-:I.- rii:ide up i i i :iii ideiit ic:il m i i i i i e i ' . The prep:ir:it i o i i of the 1:tt)elrtl iiiateri:iI l)y cJx1)o,-iiri, I ( I iritiiiiii tias been reported.& Feinale Spragiie-llawley rata e:tc,h weigliitig :ipprcisirriatel!. 2.31) g \$-ere hoiised iii individrial metaboli~m('ages designed fur tlic separation arid collection of iiriiie niid fete