sulfatroxazole: pharmacokinetics, metabolism and urinary excretion in goats and pigs
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J . vel. Phantuol. Therap. 9,5542, 1986.
Sulfatroxazole: pharmacokinetics, metabolism and urinary excretion in goats and pigs L. D. B. KINABO* & POUL NIELSEN Department of Pharmacology and Toxicology, Royal Veterinary and Agricultural University, Copenhagen, Denmark
Kinabo, L.D.B. & Nielsen, P. Sulfatroxazole: pharmacokinetics, metabolism and urinary excretion in goats and pigs. J. vel Pharmacol. Therap. 9, 55-62. The disposition of sulfatroxazole (STZ) has been studied in goats and pigs after intravenous administration of a single dose. The percentage of protein-binding decreased with increasing plasma concentration in both species. At 100 pg/ml about 85% was bound to plasma proteins in goats, while the corresponding value was only 55% in pigs. Two metabolites of STZ were isolated from urine and identified as N-acetyl-STZ and 3-sulfanilarnido-4-methyl-5-hydroxy-niethyl- isoxazole (5-OH-STZ). The goats excreted about 80% of the dose in urine. The majority (64%) was excreted as unchanged STZ, while N-acetyl-STZ and 5-OH-STZ made up 19%) and It%%#, respectively. The pigs excreted 95%, of the dose in urine. Unchanged STZ amounted to 30% and N-acetyl-STZ to 70% of the urinary excretion in pigs, while there were only traces o f 5-OH-S?Z.
Dr L. D. B . Kinabo, Department of Pharmacology and Toxicology, Royal Veterinapy mid Agricultural University, Copenhagen, Dentmark.
Recently, a new sulfonamide, sulfatroxazole (STZ), has been marketed in combination with trimethoprimt for the treatment of bacterial infections in domestic animals. The molecular structure of STZ is shown in Fig. 1 together with that of sulfamethoxazole, from which it differs only in having an additional methyl group. The figure shows further that the two compounds are also very similar to each other with respect to pK,, value and lipid solubility.
There have not yet been any reports in the literature on the pharmacokinetic parameters of STZ, and it has therefore been the purpose of the present work to study the disposition kinetics and the metabolism of this drug in goats and pigs.
MATERIALS AND METHODS
Nine experiments were performed in six clinically healthy goats (Danish Landrace) weighing 17-47 kg. They were fed with concentrate, hay and straw, and had free access to water.
Three clinically healthy animals (Danish Landrace) weighing 19-31 kg were used for this study. The pigs were fed with a coninier- cia1 fodder mixture and had free access t o water.
STZ was dissolved in water to which a few
56 L. D. B. Kinabo [3 P . Niehen
drops of 2N NaOH was added, in order to facilitate dissolution. The drug (30 mg/kg b.wt. to the goats, and 12.5 mg/kg b.wt. to the pigs) was administered to the animals by a single intravenous injection in a jugular vein in goats, and in an ear vein in pigs. About 20 blood samples were drawn from the opposite vein over the next 48 h. Plasma was separated from heparinized blood samples and stored at -20C prior to analysis.
In three experiments with goats and three with pigs, urine was collected quantitatively for 3 d. By means of a catheter, the bladder was emptied completely 6, 12, 24, 30,48, and 72 h after administration of the drug. Urine samples were stored at -20C prior to analy- sis.
The binding of STZ to plasma proteins were determined in vitro after adding the drug to plasma samples from goats and pigs as described by Nielsen 8c Rasmussen (1977). In addition, protein binding was also estimated in vim in plasma samples collected after administration of STZ to goats, as indicated in Fig. 2. Protein binding was determined by equlibrium dialysis using a Dianorm appar- atus immersed in a water bath at 37C for 90 min. Plasma samples were dialysed against an equal volume of isotonic phosphate buffer, pH 7.4, through a Visking tubing membrane with an average pore size of 2.4nm, which allows molecules with MW lower than 12,000- 14,000 to pass.
Lipid solubility was determined by shaking equal volumes of a 0.4 mM solution of STZ in
phosphate buffer, pH 7.4, and an organic solvent (chloroform or dichloroethane) at room temperature until equilibrium, as de- scribed by Nielsen & Rasmussen (1977). The results are given as the percentage of STZ in the organic solvent at equilibrium (Fig. 1).
The concentration of STZ in plasma, plasma-ultrafiltrate, and urine was deter- mined by the method of Bratton & Marshall (1939). Total sulfonamide (STZ + N'-acetyl- STZ) was determined in the same samples after acid hydrolysis, and the concentration of N'-acetyl-STZ calculated as the difference between the concentration of STZ before and after hydrolysis.
The relative amounts of unchanged STZ and of a major metabolite with a free amino group in urine was determined by thin-layer chromatography, as described by Nielsen (1973a), with chloroform-methanol (80:20, v/v) as solvent.
Isolation and identification of metabolites
Urine from goats was concentrated by evaporating about 50% of the water under reduced pressure at 37"C, after which the concentrate was subjected to gel filtration (column: lOOcm x 5 c m packed with Sephadex (325) in order to remove salts. The metabolites were further purified by means of high-performance liquid chroma- tography (HPLC). Column: 250 X 4.6mm packed with 5 pm Nucleosil C I ~ , solvent: acetonitrile-water (30:70, v/v), flow: 1 .O mumin. Retention times for the two major metabolites were 3.2 min (5'-OH-STZ) and 6.4 min (N'-acetyl-STZ). The metabolites were
NH2 Sulfomethoxoz ole
6.0 PKa Lipid solubility % 1 1 1 : chloroform 9
identified by means of mass spectrometry and H '-NMR-spectroscopy.
Electron-impact mass spectra were obtained on a VG 7070 instrument using a direct inlet at 180-200C. The ionization potential was 70 eV, and the source temperature 220C.
NMR-spectra were run on a NMR AM 500 Bruker instrument equipped with pulse/FT facilities. The samples were dissolved in deuterium oxide to which a small amount of deuterated sodium hydroxide was added.
The plasma STZ data from each animal were analysed by a non-linear curve fitting program (SAS). From this, the pharmacokin- etic model that best described the data was determined, and the actual kinetic parameters were calculated from the experimental con- stants (Baggot, 1977).
The binding of STZ to plasma proteins was dependent on the concentration of STZ in
both goats and pigs as illustrated in Fig. 2. The figure also shows that protein binding of STZ was much greater in goats than in pigs. At 100 pg/ml, 85-90% was bound to plasma proteins in goats, while the corresponding value was only about 55% in pigs.
In five out of six goats, the elimination of STZ from plasma was best described by the three-compartment open model, whereas the two-compartment open model gave the best fit to the data from one goat, and from all pigs. The distribution was a biphasic process in goats (Fig. 3a), where the mean half-lives were tl!21"f = 7 f 4 min and tl!4a = 6 9 f 4 5 min (Table I). In pigs the distribution was fast (Fig. 3b), with a mean half-life of 5 f 2 min. The elimination half-life was, on average, 366f126 min in goats, and 336f26 min in pigs (Table I).
The apparent volume of distribution (Vd area) was about 30% of the body-weight in both goats and pigs. The volume of the central compartment (V,) was 145f7 ml/kg in goats and thus approximately 50% of the total volume, while it made up about two-thirds (221 531 ml/kg) of the total volume in the pigs (Table I). The total body clearance ( C ~ B ) was about 0.6 ml/min/kg in both species (Table I).
58 L. D. B. Kinabo k? P. Nielsen
loor ( b ' pig
Time ( h 1
Identification of metabolites Similar investigations showed that urine from pigs contained unchanged STZ, a major metabolite (N'-acetyl-STZ), and also trace amounts of a metabolite with a free amino group. Identification of the major metabolites was based on the following observations.
Investigation by TLC of urine samples from goats revealed the presence of un- changed STZ, two major metabolites (N'- acetyl-STZ and 3-sulfanilamido-4-methyl-5- hydroxymetbyl-isoxazole = 5'-OH-STZ) and a minor fraction of conjugated metabolites. NJ-acetyl-STZ. This compound did not react
Suljialroxazok in goats mid pigs 59
TABLE I . Pharmacokinetic parameters describing disposition of sulfatrox- azole in goats (n=5) and pigs (n=3) after intravenous injection o f a single
Goats Pigs Parameter Units (Mean + SD) (Mean f SD)
30 2 0 8 f 10 5 7 + 14
0. I395 f 0.0869 7 f 4
5 2 + 17 0.0131 f 0.0061
69 f 45 9 9 f 15
0.00208 f 0.00069 366+ 126 145 f 7 271 f 18
59,000 + 200 0.57 k 0.20
3 0 + 13
12.5 57 f 8
1 6 f 4 0.1689 * 0.0690
5 + 2 41 2 4
0.00207 f 0.00015 336 f 26 221 + 31 303 f 30
20,000 f 600 0.63 + 0.02
2 6 f 6
with p-dimethylamino-benzaldehyde, suggest- ing that a substituted "-amino group and its chromatographic and mass spectral data were identical to those of synthetic reference material. The mass spectrum revealed ions at m/z 309 M+, 245 [M-SO$, 198, 175, 157, 135, 108, and 92.
5 -OH-STZ. This metabolite reacted with p-dimethyl-amino-benzaldehyde, indicating a free N4-amino group. TLC and HPLC showed that the metabolite was more polar than STZ. Sulfanilamide was slowly released on heating the metabolite in the mass spectro- meter. Th