SHORT COMMUNICATION J . vet. Pharmatol. Therap. 12,459-462, 1989.

Lack of oxidative pathways in the metabolism of sulphisomidine by the turtle Pseudemys scripta elegans T. B. VREE,*t J . B. VREE,$ P. A. M. DE JONGE,* M. J. M. PLAUM,* C. P. W. G. M. VERWEY,* Y. A. HEKSTER,* M. SHIMODA,§ & J . F. M. NOUWSn

Departments of *Clinical Pharmacy and tAnesthesiology, Sint Radboud Hospital, Geert Grooteplein Zuid 8, Nijmegen, SDotulabs, Weezenhof 35-08, Nijmegen, IR.V.V.-kring 6, Wolfskuilseweg 279, Nijmegen, the Netherlands, §Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan

Sulphadimidine (2-sulfanilamido-4,6-di- methylpyrimidine) and sulphisomidine (4- sulfanilamido-2,6-dimethylpyrimidine) are structural analogues, which are subject to different metabolic pathways in man. For instance, sulphadimidine is predominantly N4-acetylated (SO-SO% of the dose), and ex- hibits a ‘fast-slow’ acetylator phenotype, while only 30% of a dose of sulphisomidine is unimodally acetylated (Vree et al., 1985, 1986~).

In ruminants, sulphadimidine is minimally acetylated, and mainly oxidized at the 4- methyl group, forming 4-hydroxy- and even 4-carboxysulphadimidine (Nouws et al., 1987a,b, 1988a); these oxidation pathways account for merely 10-2076 of the dose in man (Vree et al., 1986b).

The mass balance (percentage of the dose recovered) of sulphisomidine in man (S+N4)

is almost loo%, leaving little opportunity for oxidative pathways. Oxidation of sulphisomi- dine in animals has not yet been reported. Fresh-water turtles are able to oxidize sulpha- dimidine (Vree et al., 1986a) and the high- performance liquid chromatography (HPLC) chromatograms of their cumulative excretion products are devoid of endogenous and inter- fering compounds (Vree et al., 1989). Hence, if hydroxy metabolites of sulphisomidine were formed in animals, the detection of these products should be demonstrated in the turtle as were they for sulphadimethoxine (Vree et al., 1989) and sulphadirnidine (Vree el al., 1986a).

The aim of this investigation was to assess whether the methyl substituents in 4- sulphanilamido-pyrimidines can be oxidized into corresponding hydroxy or even carboxy metabolites as illustrated in Fig. 1. For this purpose, sulphisomidine was selected as test drug and the turtle Pseudemys scripta elegans as the animal species used.

Four turtles, deprived of all food for 2 weeks with 500 k 108 (SD) g body weight were obtained from Dotulabs. The turtles were kept in a 90 X 25 X 40-cm aquarium filled with 25 1 of water of 23°C. A sulphisomi- dine dose of 247 mg was mixed with liver sausage, divided into small portions, and fed to the four turtles. Two 2-ml samples were taken immediately from the tank and the water was changed (25 I volume, 23°C). Thereafter 2-ml water samples from the tank were taken over a period of 200 h for the analysis of excreted sulphisomidine and its metabolites. The tank samples were kept at -20°C until analysis. The tank was lighted from 09.00 h until 21.00 h with a 30-W UV light.

Sulphisomidine and its metabolites were analysed by high-performance liquid chroma- tography. The column was Spherisorb ODS, 5 WM, 250 x 4.6 mm ID (Chrompack, Middelburg, the Netherlands) with a guard column 50 X 4.6 mm, 10 WM. The mobile phase was a mixture of 2.44 g of sodium acetate, 60 ml of acetonitrile, 70 ml of metha- nol and 5.7 ml of 0.2 moYl acetic acid in 1 I of water. Flow rate was 1.2 ml/min. Ultra-

459

460 T. B. Vree et al.

ocetylot ion

4

Sulf isomidine

I CH20H

FIG. 1. Metabolic scheme of sulphisomidine and its tentative metabolites.

violet detection was achieved at 272 nm. The capacity factors k' (capacity factor k' = &-to)/ to; with t, the retention time of the solute, and to the retention time of the unretained) of sulphisomidine and its anticipated metabolites are shown in Table I.

Deglucuronidation was carried out in three different ways: a 1-ml sample was mixed with 0.5 ml of 0.2 moUl KHzP04 and 0.1 ml of p glucuronidase (E. coli, pH 6.8, 10 000 u/ml; H e h pomatia type H2 10 000 u/ml, pH 5.0; type BI 10 000 u/ml, pH 5.0; Sigma, St Louis, MO). The mixtures were allowed to react for 6 h at 37°C. Desulphation: a 1-ml sample was mixed with 0.5 ml of 0.2 moYl sodium acetate (pH 5.0) and 0.1 ml of arylsulphatase (pH 5.0; 200 u/ml; Sigma, St Louis, MO). The efficien- cy of both deconjugation reactions was tested with a sample of human urine containing sulphadimethoxine and its glucuronidated metabolite.

Drugs

Sulphisomidine was obtained from Sigma (St Louis, MO) and N4-acetylsulphisornidine was synthesized (Vree el al., 1985; Vree & Hekster 1987).

The metabolites 4-hydroxy-, 6-hydroxy-, 4-carboxy-, 6-carboxy-, Nh-acetyl-4-hydroxy-, N4-acetyl-6-hydroxy-, N4-acetyl-4-carboxy-, and N4-acetyl-6-carboxysulphisomidine were synthesized by the oxidation of N4- acetylsulphisomidine with selenium dioxide in dioxane until the corresponding aldehydes and carboxy acids were formed. The alde- hydes were reduced with potassium boro- hydride in methanol to the corresponding alcohols. Deacetylation was carried out in 1 mol/l HCI. Molecular weights of the synthe- sized compounds were confirmed by fast- atom-bombardment mass spectrometry. NMR spectra were recorded with a Varian EM 390 spectrophotometer at 9OMHz.

The HPLC chromatograms of the tank- water samples showed only the presence of sulphisomidine and N4-acetylsulphisornidine after the administration and excretion of 247 mg of sulphisomidine to the four turtles. No hydroxy or carboxy metabolites of sulphiso- midine and its corresponding conjugates were detected. Figure 2 shows the cumulative excretion-time curves of sulphisomidine and its metabolite N4-acetylsulphisomidine. At the end of the sampling period, 25% of the dose of sulphisomidine was recovered, while the

Sulphisomidine metabolism in the turtle 461

TABLE I. Capacity factors of sulphisomidine and its metabolites

Capacity factor Ratio

Compound Reference compounds Turtle samples N41S 60H/20H

- - 2-OH-sulphisomidine 1.54 - 6-OH-sulphisornidine 2.00 - 6-carboxysulphisornidine 2.54 - Sulphisomidine 2.62 2.62 - - N4-acetyl-2-OH-sulphisomidine 3.38 - 2.19 - N4-acet yl-6-OH-sulphisomidine 3.76 - N4-acetyl-6-carboxysulp hisomidine 4.23 - 1.67 - N4-acetylsulphisomidine 5.69 5.69 2.17 -

1.30 - - -

1.88 1.25

Ratio = ratio between the capacity factors, N4 = N4-acetylsulphisomidine, S = sulphisomidine, 2 0 H =

Capacity factor k' = (tr-to)/lo; 1, retention time of the solute, and to retention time of the unretained. 2-hydroxysulphisomidine, and 6 0 H = 6-hydroxysuIphisomidine.

100 I

I I I I I 0 50 100 150 200 250

Time f h )

FIG. 2. Cumulative excretion-time profile of 25.1 % sulphisomidine ( 0 ) and its metabolite N4- acetylsulphisomidine (0) by four turtles Pseudemys scripla ehgans after administration of an oral dose of 247 mg of sulphisomidine.

N4-acetyl metabolite disappeared from the tank.

The position of the nitrogen atoms in the N1-pyrimidine substituent of the sulphona- mide analogues sulphisomidine and sulphadi- midine determines the susceptibility of the methyl groups for oxidation. The turtle is able to oxidize the methyl groups of sulphadimi-

dine (Vree et al . , 1986c), as are other species such as ruminants, birds, fish, and snails (Nouws et al., 1986, 1988b; Vree et al . , 1986~; Geertsma et al., 1987). Humans oxidize sul- phadimidine only in minor amounts (Vree et al., 1986b). In humans sulphisomidine is acetylated, and the mass balance of parent drug and metabolite amounts to loo%, leav- ing no opportunity for oxidation. This study shows that the turtle is not able to oxidize sulphisomidine. It must be concluded that the methyl groups in sulphisomidine are not accessible for oxidation.

The cumulative excretion-time curve of the N4-acetyl metabolites shows an unexplained phenomenon. The compound is formed in and excreted b y the turtles. After 100 h, hydrolysation of N4-acetyl sulphisomidine into sulphisomidine, resulting from the UV light in the tank, commences. A similar hydrolysis phenomenon was observed with N4-acetylsulphamethoxazole, whether it was present as metabolite, or administered as parent drug to the turtles. When the com- pound was added to the tank water, it was stable for 200 h, and thereafter hydrolysed to sulphamethoxazole. Hydrolysis is prevented when the experiment is conducted in the dark (Vree & Vree 1983; Vree et al., 1987). The question as to why this hydrolysis only starts aft& a period of 100 h is not answered by the results of this or the previous study (Vree et al., 1987).

462 T. B . Vree et al.

A C K N O W L E D G M E N T S

Dr R. Fokkens, and Professor N. M. M. Nibbering, Institute for Mass Spectrometry, University of Amsterdam are thanked for recording the FAB mass spectra of sulphi- somidine and the synthesized hydroxy deriva- tives.

R E F E R E N C E S

Geertsma, M.F., Nouws, J.F.M., Grondel, J.L., Aerts, M.M.L., Vree, T.B. & Kan, C.A. (1987) Residues of sulphadimidine and its metabolites in eggs following oral sulphadimidine medication of hens. Veterinaty Quarterly, 9, 67-75.

Nouws, J.F.M., Vree, T.B., Breukink, H.J., van Miert, A.S.J.P.A.M. & Grondel, J.L. (1986) Phar- macokinetics, hydroxylation and acetylation of sulphadimidine in mammals, birds, fish, reptiles and mollusks. In Comparative Veterinary Phannacol- ogy, Toxicology and Therapy. Eds A.S.J.P.A.M. van Miert, M.G. Bogaert & M. Debackere. pp. 415- 426. MTP Press, Lancaster.

Nouws, J.F.M., Firth, E.C., Vree, T.B. & Baakman, M. (1987a) Pharmacokinetics and renal clearance of sulfamethazine, sulfamerazine and sulfadia- zine and their N4-acetyl and hydroxy metabolites in horses. A m ' c a n Journal of VeterinaT Research, 48, 392402.

Nouws, J.F.M., van Miert, A.S.J.P.A.M., van Gogh, H., Watson, A.D.J. & Vree, T.B. (1987b) The effect of tick-borne fever on the metabolism and renal clearance of sulfadimidine in goats. Pharmu- ceutisch Weekblad (Scientific Edition), 9, 91-98.

Nouws, J.F.M., Geertsma, M.F., Grondel, J.L., Aerts, M.M.L., Vree, T.B. & Kan, C.A. (1988a) Plasma disposition and renal clearance of sulpha-

dimidine and its metabolites in laying hens. Research in Veterinaty Science, 44, 202-207.

Nouws, J.F.M., Meesen, B.P.W., van Gogh, H., Korstanje, C., van Miert, A.S.J.P.A.M., Vree, T.B. & Degen, M. (1988b) The effect of testoster- one and rutting on the metabolism and pharma- cokinetics of sulphadimidine in goats. Journal of Veterinury Pharmacology and Therapeutics, 11, 145- 154.

Vree, T.B. & Vree, J.B. (1983) Acetylation of sulphamethoxazole by fresh water turtles Pseudays scripta ekgans. Jounull of Vetennay Phar- macology and Therapeutiu, 6 , 237-240.

Vree, T.B., Hekster, Y.A. & Tijhuis, M.W. (1985) Metaholism of sulfonamides. Antibiotics and Chpmotherapy, 34, 66-120.

Vree, T.B., Vree, J.B. & Nouws, J.F.M. (1986a) Acetylation and hydroxylation of sulfadimidine by the turtle Cuora amboniemis. J o u m l of Veterin- ary Pharmacology and Therapeutics, 9, 330-332.

Vree, T.B., Hekster, Y.A., Nouws, J.F.M. & Baak- man, M. (1986b) Pharmacokinetics, metabolism, and renal excretion of sulfadimidine and its N4- acetyl and hydroxy metabolites in humans. Therapeutic Drug Monitoring, 8, 434439.

Vree, T.B., Vree, M.L. & Nouws, J.F.M. (1986~) Acetylation and hydroxylation of sulphadimidine in the snail Cepaea hortmis. Journal of Veterinary Medicine A, 33, 633-636.

Vree, T.B. & Hekster, Y.A. (1987) Clinical pharma- cokinetics of sulfonamides and their metabolites. Antibiotics and Chemotherapy, 37, 25-30.

Vree, T.B., Vree, J.B., Hekster, Y.A. & Nouws, J.F.M. (1987) Acetylation, deacetylation and hy- droxylation of sulphamethoxazole and N4- acetylsulfamethoxazole in the turtle Pseudemys scripta ekgans. Veterinary Quarterly. 9, 381-384.

Vree, T.B., Vree, J.B., Beneken Kolmer, N. et al. (1989) 0-demethylation and N4-acetylation of sulfadimethoxine by the turtle Pseudemys scfipta ekgans. Veterinary Quarterly, 11, 138-143.