Pyridinethione glucuronide as a metabolite of sodium pyridinethione

Fd Cosmet. Toxicol. Vol. 9, pp. 519-526. Pergamon Press 1971. Printed in Great Britain

Pyridinethione Glucuronide as a Metabolite of Sodium Pyridinethione B. L. KABACOFF,C. M. FAIRCHILD and C. BURNETT Division of Basic Research, Revlon Research Center, 945 Zerega Avenue, Bronx, New York 10473, USA (Received4 February 1971)

Abstract--Urine from rats given a single oral dose of sodium pyridinethione (SPT) contains a material which, when separated by thin-layer chromatography, has an ultraviolet absorption spectrum similar to that of a group of pyridine-l-oxides substituted in position 2. An SPT metabolite with the same spectral characteristics was isolated from rabbit urine after intravenous administration of SPT. Isolation of sufficient metabolite for characterization was accomplished by passage of this urine first through Sephadex G10 gel, and then through Dowex 50 ion-exchange resin. The purified metabolite gave positive tests for glueuronic acid and yielded pyridinethione on hydrolysis with/~-glucuronidase. Elemental analysis for C, H, N, O and S corresponded to theory for pyridinethiol-l-oxide giucuronide. INTRODUCTION It has previously been reported in this journal (Min, Parekh, Golberg & McChesney, 1970) that when sodium pyridine-2-thiol 1-oxide (sodium pyridinethione; SPT) is applied to rat skin, the major metabolite is pyridine-N-oxide 2-sulphonic acid. The excretion of traces of pyridinethione and pyridinethione disulphide was also observed. The experiments described below establish that when SPT is administered intravenously to rabbits, a significant amount is excreted as the glucuronide. EXPERIMENTAL Materials. The SPT used in these experiments was obtained from the Olin Chemical Co., New York. Phenolphthalein glucuronide, sodium glucuronate and fl-glucuronidase used in the identification of the metabolite were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. The fluorescent thin-layer chromatography plates used were Eastman silica gel 6060 and 6065 cellulose (Distillation Products Industries, Rochester, N.Y.), and Brinkmann silica gel F254 and alumina Alox N-HR/UV-254 (Brinkmann Instruments Inc., Westbury, N.Y.). Visualization of ultraviolet-absorbing materials on these plates was made possible by the use of the UVS 12 lamp fUltra Violet Products Inc., San Gabriel, Cal.). Isolation and purification of the metabolite was accomplished by the use of Sephadex LH20 and G10 (Pharmacia Fine Chemicals, Piscataway, New Market, New Jersey) and Dowex 50X12 (200-400 mesh) (Bio Rad Laboratories, New York). Isolation and purification of the glucuronide from rats. Rats were given an oral dose of 100 mg SPT/kg and urine was collected over a period of 3 hr. A fluorescent silica-gel thinlayer chromatography sheet (Eastman 6060) was spotted with this urine and with urine

519

520

B.L.

KABACOFF, C. M. FAIRCHILD

and

C. BURNETT

from control rats. The chromatogram was then developed with methanol and observed under ultraviolet light (254 nm). Gel filtration was used to obtain larger quantities of an ultraviolet-absorbing substance present on the chromatogram of the test urine but not present in control urine. A 2.5-ml aliquot of each urine sample was added to 25 ml methanol to precipitate salts. The filtrates were evaporated to dryness in vacuo at 38°C and taken up in 2-5 ml methanol. The methanolsoluble material was chromatographed with methanol on a 2.5 x 48-cm column of Sephadex LH 20. In all studies, 5-ml fractions were collected. Experiments with rabbits Isolation and purification o f metabolite. On the assumption that they would produce the same SPT metabolite as did the rats, rabbits were used to provide sufficient metabolite for the characterization of its structure. In a typical experiment demonstrating the final method of isolating and purifying the metabolite, rabbits weighing about 2 kg were dosed intravenously with 100 mg SPT/kg and urine was collected for 3 hr thereafter. After adjustment to pH 3.7 and centrifugation, the volume of the supernatant was 130 ml. This material was taken to dryness in vacuo and the residue was taken up in 10 ml water. The water-soluble portion was chromatographed by ascending technique in water on a 2.5 x 36-cm Sephadex G I 0 column. The fractions giving an absorption peak around 234 nm were pooled, concentrated to 10 ml in vacuo and passed through a 3.0 x 16-cm column of Dowex 50 x 12, in the acid form. The column was eluted with water. The colourless fractions having an absorption peak at 234 nm were pooled and concentrated to a glassy residue in vacuo. The residue was washed out of the evaporator flask with a minimum amount of anhydrous methanol. A white crystalline material was collected on a Buchner funnel and dried at 60°C in vacuo. In one case, the metabolite was recrystallized from methanol-ethylene dichloride. Charaeterization as the glucuronide. Five ,~ spots of a 2.2 ~o solution of the metabolite were chromatographed to a distance of 10 cm on a Brinkmann F254 silica-gel plate in methanol. The chromatograms were observed under ultraviolet light (254 nm), sprayed with 50 ~o aqueous sulphuric acid and heated at 105°C, and treated with naphthoresorcinol to detect uronic acids (Stahl, 1965). The Tollens test for glucuronides and glucuronic acid (Hawk, Oser & Summerson, 1947) was run on phenolphthalein glucuronide, sodium glucuronate and the metabolite. Infra-red study. The infra-red spectrum of the metabolite was determined on 0"3~o solutions in KBr and in dioxane, using the Perkin-Elmer 621 Grating IR Spectrophotometer (Perkin Elmer, Norwalk, Conn.). Enzymatic hydrolysis. A 2~o solution of/3-glucuronidase was prepared in 0"1 N-acetate buffer at pH 4.7, and 10-ml aliquots of the enzyme solution were added to 10 ml of a 0.1 ~o aqueous solution of the metabolite, to 10 ml of a 0"05~o aqueous solution of SPT and to 10 ml distilled water (enzyme blank). The three solutions were incubated for 24 hr at 37°C and were then extracted with three 10-ml portions of chloroform. The absorption spectra of the chloroform extracts of the metabolite and the SPT were determined against the chloroform extract of the enzyme blank. Portions of the chloroform extracts were evaporated to dryness in vacuo at room temperature, the residues were dissolved in 0-001 N-HCI and in 0.001 r~-NaOH, and absorption spectra were determined as above. Elemental analyses. Analyses were conducted to support the proposed glucuronide structure of the SPT metabolite and to monitor its purity. The F. and M. Model 185

SODIUM PYRIDINETHIONEMETABOLITE

521

Semiautomatic C H N Analyzer (F. and M. Scientific Corp., Avondale, Penn.) was used for the carbon, hydrogen and nitrogen determinations, the elements being converted to CO2, H 2 0 and N2, respectively, and automatically determined by gas-liquid chromatography. Sulphur was determined by oxidation to sulphate followed by precipitation of the sulphate as the lead salt (Schwarzkopf & Schwarzkopf, 1969). In the determination of oxygen, the sample was pyrolysed to form various oxides, which were then converted to CO by heating with carbon. The CO was then oxidized to CO2 (Schwarzkopf & Schwarzkopf, 1969). RESULTS Isolation and purification o f glucuronide f r o m rats

The RF values of the ultraviolet-absorbing materials isolated by thin-layer chromatography from the urines of rats treated with SPT and of controls are given in Table 1. The material in the test urine with an Rr value of 0.53 was evidently a normal component, Table 1. Rv values of ultraviolet-absorbing materials from the urine of control rats and of rats given an oral dose of SPT

Source of urine Control rats Treated rats

Rv -4- SD 0.54 4- 0"05 0-36 4- 0"03 0.53 4- 0"02

because a similar spot was obtained with control urine. The urine from treated rats also contained a substance with an RF value of 0.36. When urine from treated rats was chromatographed on a Sephadex column, fractions from 115 to 140 ml showed peak absorbances at about 238 and 265 nm and a shoulder at 300-310 nm. No similar fractions were found with the control urine. The fractions of control urine with the highest ultraviolet absorbance were those taken between 125 and 165 ml. These, like the 115-140-ml fractions of the test urine, were pooled and concentrated in vacuo for thin-layer chromatography, and the migration rates of the ultraviolet absorbing components are given in Table 2. Except for the material of R~ 0.68, the RF values were the same as those obtained by thin-layer chromatography of the original urines. The material with an _Rp value of 0.38 was eluted from the chromatogram with methanol. The absorption spectrum in the same solvent is given in Fig. 1, and it is noteworthy that the absorption spectrum is similar to that of pyridine-l-oxides substituted in position 2, as Table 2. R~ values of ultraviolet-absorbing materials after Sephadex chromatography of urine samples from control rats and from rats given an oral dose of SPT

Source of urine Control rats Treated rats

Re + SD

0.56 :tz 0-05 0"38 ~ 0-02 0.58 ~ 0"03

0.68 + 0"02

522

B.L.

KABACOFF,

C. M.

FAIRCHILD

and

c.

BURNETT

o= o

200

250

300 Wavelength,

350

400

nm

FIG. 1. Absorption spectrum of material with an Rr value of 0"38 from urine of rats given an oral dose of SPT. shown in Table 3. This similarity to known 2-substituted p y r i d i n e - l - o x i d e s indicates that the material with an RF value o f 0.38 was a derivative o f SPT. The a b s o r p t i o n spectrum o f the material with an RF value o f 0.68 is given in Fig. 2. It a p p e a r e d neither in the course o f thin-layer c h r o m a t o g r a p h y o f urine before Sephadex Table 3. Absorption maxima of pyridine-l-oxides substituted in position 2

Substance

Absorption maxima (Wavelength and relative intensity*)

Material with Rr 0"38 238 (1"0) 2-Benzylthiopyridine-l-oxidel" 238 (1.0) 2-Acetamidopyridine-l-oxidet 233 (I.0) 2-Benzyloxypyridine-l-oxide~. Not given

262 (0.35) 262 (0-36) 262 (0.36) 260 - -

300 (0-11) 295 (0-16) 295 (0.13) 304 - -

*The absorbance relative to that of the peak at the lowest wavelength. t Jones & Katritzky (1960). +Shaw (1949). c h r o m a t o g r a p h y n o r in the column eluates and was only in evidence after thin-layer c h r o m a t o g r a p h y o f the pooled, concentrated column eluates. These observations suggest that this material was either a m i n o r constituent or a b r e a k d o w n p r o d u c t . In any case further study o f this material was deferred. Experiments with rabbits The a b s o r p t i o n spectra o f the fractions o b t a i n e d by the c h r o m a t o g r a p h y o f the r a b b i t urines on Sephadex a n d Dowex columns were very similar to those o f the r a t metabolite. Thin-layer c h r o m a t o g r a p h y o f the metabolite crystallized from these fractions isolated ultraviolet-absorbing material with an RF value o f 0-62. Spraying with 50 ~o aqueous sulphuric acid and heating at 105°C again revealed only one spot ( R r 0.62), which also gave a

SODIUM P Y R I D I N E T H I O N E

METABOLITE

523

0.6

O-4

g

ao 0.2

o

200

250 Wavelength,

I

300 nrn

FIO. 2. Absorption spectrum of material with an Rf value of 0.68 from Sephadex-chromatographed urine of rats given an oral close of SPT.

positive reaction for uronic acids. In the Tollens test, phenolphthalein glucuronide, sodium glucuronate and the metabolite all showed the same absorption spectrum with a peak of 570 nm. It was concluded that the metabolite contained a glucuronic acid moiety. In the infra-red study, the metabolite showed a peak at 3380 cm-1 in KBr and at 3320 cm- t in dioxane. When the metabolite and SPT were treated enzymatically to split off the aglycone portion of the molecules, the absorption spectra of chloroform extracts of the two hydrolysates were found to be the same between 250 and 400 nm (Fig. 3). Similarly it was evident from the absorption spectra of residues from the evaporated chloroform extracts redissolved in HCI and NaOH that the hydrolysed metabolite and SPT were identical (Figs 4 & 5). When the chloroform extracts of hydrolysed metabolite and SPT were compared by thin-layer chromatography using methanol as the developing solvent, both had an Rf value of 0.59 on Eastman 6065 cellulose and of 0-04 on alumina (Alox N-HR/UV-254, Brinkmann). Spraying the chromatoplates with 0-1 ~o ferric chloride produced a violet colour with both materials, presumably due to the formation of the ferric chelate. This reaction did not occur with the unhydrolysed metabolite.

°T /", /

\

f,. 250

I

I

300

:350

Wavelength,

FIo. 3. Absorption spectra of glucuronidas¢ hydrolysate,s of SPT ( (- - -) in c h l o r o f o r m . F.C.T. 9/4 e

400

nrn ) and pyridinethione metabolite

524

a.L.

KABACOFF, C. M. FAIRCHILD

and c. BURNETT

'~ 0 ' 5

o

J

240

I ~='~

250

I

550

300

Wavelength,

~'~I 400

nm

FIG. 4. Absorption spectra of glucuronidase hydrolysates of SPT (

) and pyridinethione metabolite

( - - - ) in 0-00] N-HCI.

I

I.O

P

<

0~ '"

240

~\ I

250

300

Wavelength,

350

FiG. 5. Absorption spectra of glucuronidase hydrolysates of SPT (

(---) in 0.001 N-NaOH.

f

400

nm

) and pyridinethionemetabolite

These findings indicate that the structure of the metabolite contains both SPT and glucuronic acid. The formula of the S-glucuronide would be C1 ,H13NOTS (mol wt 303"3), and this structure was supported by the elemental analyses carried out on the metabolite (found: C, 43.89; H, 4-55; N, 4"60; S, 10.94; O, 35"95; CIIHI3NOTS requires C, 43.57; H, 4.32; N, 4-62; S, 10.57; O, 36.93 ~). DISCUSSION Thiols, phenols, alcohols, carbohydrates, carboxylic acids and amines are known to be metabolized by conjugation with glucuronic acid (Dutton, 1962). Although it is unlikely that the glucuronide bond is linked to the aminoxide group, this possibility will be dis-

SODIUM PYRIDINETHIONE METABOLITE

525

cussed below. It has been shown in this paper that the aglycone p o r t i o n of the metabolite is pyridine-2-thiol I-oxide. As already mentioned, pyridine-N-oxides, substituted in position 2 with an acetamido, benzyloxy or benzylthio group, have an a b s o r p t i o n b a n d at 260 n m . When the oxygen atom of the aminoxide group carries a substituent, as in N-benzyloxy-2pyridone, the 260 n m b a n d disappears (Shaw, 1949). The 260 n m b a n d is present in the metabolite obtained from SPT. In the infra-red region, the metabolite shows a peak at 3380 c m - 1 in K B r a n d at 3320 c m - 1 in dioxane. A r o m a t i c aminoxides that are h y d r o g e n - b o n d e d as they would be in the glucuronide are k n o w n to show a peak a r o u n d 3360 cm -1 (Nakanishi, 1962). Peaks in this region are not f o u n d in the spectrum of pyridine-2-thiol I-oxide. Since glucuronides formed in the animal body are/3-D-glucopyranosiduronic acids ( D u t t o n , 1962), the metabolite must be the S-/3-D-glucopyranosiduronic acid of pyridine-2-thiol 1-oxide. In this work there was very little evidence of ultraviolet-absorbing materials other than the glucuronide. O n the other hand, M i n et al. (1970) did not report the presence of a glucuronide. Possibly these differences in the excreted products derived from SPT arose from the use of different routes of administration. Acknowledgements--The authors are grateful to Dr. I. Margolin of the Revlon Research Center, who performed the carbon, hydrogen and nitrogen determinations, and to the staff of the Schwarzkopf Microanalytical Laboratory, Woodside, N.Y. for the sulphur and oxygen determinations.

REFERENCES Dutton, (3. D. (1962). (31ucuronide conjugation. In Metabolic Factors Controlling Duration of Drug Action. Edited by B. B. Brodie and E. (3. Erdos. p. 39. Macmillan, New York. Hawk, P. B., Oser, B. L. & Summerson, W. H. (1947). Practical Physiological Chemistry. 13th ed. p. 843. Mc(3raw-Hill, New York. Jones, R. A. & Katritzky, A. R. (1950). N-oxides and related compounds. Part XVII. The tautomerism of mercapto- and acylamino-pyridine 1-oxides. J. chem. Soc. p. 2937. Min, B. H., Parekh, C., (3olberg, L. & McChesney, E. W. (1970). Experimental studies of sodium pyridinethione. II. Urinary excretion following topical application to rats and monkeys. Fd Cosmet. Toxicol. 8, 161. Nakanishi, K. (1962). Infrared Absorption Spectroscopy--Practical. p. 51. Holden-Day, San Francisco. Schwarzkopf, O. & Schwarzkopf, F. (1969). Chemical characterization of organometallic compounds. In Characterization of Organometallic Compounds. Edited by M. Tsutui. p. 55. Interscience, New York. Shaw, E. (1949). Analogs of aspergillic acid. I. Tautomerism of the hydroxypyridine-N-oxides. J. Am. chem. Soc. 71, 67. Stahl, E. (1965). Thin Layer Chromatography. p. 489. Academic Press, New York.

Le glycuronide de la pyridinethione, m~tabolite de la pyridinethione de sodium R~am~---L'urine des rats qui ont re~u une dose orale unique de pyridinethione de sodium (SPT) contient un mat~riau dont le spectre d'absorption ultraviolet est similaire, quand on le s~pare par chromatographic en couche mince, ~. celui d'un groupe de pyridine-l-oxydes substitu~s en position 2. Un m~tabolite du SPT pr~sentant les m~mes caract~ristiques spectrales a ~t(~isol~ de i'urine de lapins auxquels on avait administr6 du SPT par voie intraveineuse. On a pu isoler une quantit6 de ce m~tabolite, suffisante pour l'identifler, en faisant passer l'urine d'abord sur gel Sephadex G 10, puis sur r~sine b,changeuse d'ions Dowex 50. Le m~tabolite purifi6 a r~agi positivement aux tests de racide glycuronique et son hydrolyse par la ~-glycuronidase a donn6 de la pyridinethione. Les r~ultats de l'analyse ~16mentairede C, H, N, Oet S ont concord6 avec l'hypoth~e du glycuronide du pyridinethiol-l-oxyde.

526

B . L . KABACOFF, C. M. FAIRCHILD and c. BURNETT Pyridinthionglucuronid als M e t a b o l i t von N a t r i u m p y r i d i n t h i o n Zusammeafassung--Urin yon Ratten, die eine einzelne orale Dosis von Natriumpyridinthion (SPT) erhielten, enth~.lt ein Material, das nach Abtrennung mittels Di~nnschichtchromatogra phie ein ultraviolettes Absorptionsspektrum ~ihnlich dem einer Gruppe von Pyridin-l-oxyden, die in Position 2 substituiert sind, ergibt. Ein SPT-Metabolit mit den gleichen spektralen Eigensehaften wurde nach intraven~ser Anwendung yon SPT aus Kaninchenurin isoliert. Die Isolierung einer ffir die Charakterisierung gen[igenden Menge Metabolit wurde dadurch bewirkt, dass dieser Urin zuerst durch das Gel Sephadex G l0 und dann dutch das Ionenaustauscherharz Dowex 50 geleitet wurde. Der gereinigte Metabolit ergab positive Testergebnisse fCtr Glucurons~ure und lieferte bei Hydrolyse mit/LGlucuronidase Pyridinthion. Die Elementaranalyse auf C, H, N, O und S entsprach der Theorie f~r Pyridinthiol-l-oxidglucuronid.