Sulfatroxazole: pharmacokinetics, metabolism and urinary excretion in goats and pigs

Download Sulfatroxazole: pharmacokinetics, metabolism and urinary excretion in goats and pigs

Post on 27-Sep-2016




1 download

Embed Size (px)


  • 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.



    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.

    Experimental procedure

    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.

    Analytical methods

    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

    dichloroethane 20

    Sulfotroxozole 5.8

    12 25

  • 25 t

    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.

    Phanna.cokinetic analysis

    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).


    Protein binding

    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. The mass spectrum showed that no ions at m/z higher than 172 (sulfanilamide) were present. The slow release of sulfanil- aniide poirtted to degradation of the metabo- lite, possibly due to salt formation and corresponding low volatility. Due to this the metabolite could not be identified from its mass spectrum. However, comparing the NMR-spectrum of the metabolite with that of STZ showed that the only significant differ- ence between the two spectra was replacement of the CHS-signal at 1.95 p.p.m. with a

    CH2-signal at 4.28 p.p.m. This corresponds to the presence of a CH20H-group at position 5 of the isoxazole ring (Fig. 1).

    Urinary excretion of STZ and i h metabolites

    The goats excreted more than 70% of the administered dose within 24 h of administra- tion (Fig. 4a). On average, for three experi- ments 79+12% of the dose was recovered in urine. Unchanged STZ made up 64+8% of the amount excreted in urine, while N'-acetyl- STZ amounted to 19+4% and 5'-OH-STZ to 18+9%

    The pigs excreted just over 80% of the dose within 24 h after the administration and in total 9 5 f 9 % was recovered in urine (Fig. 4b). About 30% of the amount excreted in urine was unchanged drug, while the majority, nearly 70%, was made up by N4-acetyl-STZ. As well as these two compounds, trace amounts of a metabolite with chrornato- graphic properties similar to those of 5'-OH- STZ was present in the urine.

  • 60 L. D. B. Kinabo &f P. Nielsen

    12 24 48 72 Time ( h )

    FIG. 4. Cumulative excretion of sulfatroxazole (STZ) and its metabolites in urine after intravenous administration to (a) goats and (b) pigs. The symbols are ( 0 ) unchanged STZ; (0) N-acetyl-STZ; (A) 5-OH-STZ; (m) total urinary excretion. The bars indicate 1 SD.


    The plasma protein-binding of STZ in goats was dependent on the concentration, but it was found to be the same in samples to which STZ was added and in samples obtained after administration of the drug to goats. The binding of STZ to plasma proteins in goats exceeded 80% for plasma concentrations lower than 250 pg/ml and may be described as extensive (Craig & Welling, 1977). It is much higher than for sulfamethoxazole (54% bound at 100 pg/ml, Jfirgensen & Rasmussen, 1972), although these two compounds are structurally related and have similar lipid solubilities (Fig. 1). The pigs showed a similar dependence between protein binding and plasma concentration of STZ, as found in goats. However, the protein binding in pigs was much lower than in goats. Similar or even more pronounced species differences in pro- tein binding have been demonstrated for both acidic drugs, such as salicylate, (Sturman &

    Smith, 1967), and for basic drugs, like desi- pramine (BorgP el al., 1968).

    The distribution of STZ was faster in pigs than in goats (Table I), and in the latter species the distribution phase could be divided into two processes, reflecting differ- ences in the rate of distribution to different tissues. However, the elimination rate for STZ was approximately the same (C1~-0.6 ml/min/ kg) in the two species.

    In spite of the observed difference in plasma protein-binding between goats and pigs, the apparent volume of distribution was nearly the same - approximately 30% of the body-weight - and of the same size as reported for sulfamethoxazole in cows (Nielsen & Rasmussen 1977).

    About 80% of the administered dose was recovered in urine from goats, while the corresponding figure was 95% for pigs. Separation of STZ and its metabolites in urine showed that about 65% of the total sulfon- amide content in goat urine was unchanged

  • S u ~ ~ i h x u z o l e in ~ ( H C L V und pigs 61

    trimethoprim. From a pharmacokinetic point of view it is considered desirable to have an equal half-life for the components of com- bined drugs. In order to obtain administra- tion intervals of reasonable length it could consequently be an advantage in veterinary practice if a trimethoprim analogue with a longer half-life could be found. Although Bushby (1980) states that pharmacokinetic matching, and the ratio at which trimetho- prim and sulfonamide are administered, are seldom important because identical effects on the bacteria occur over a wide range of ratios, a synergistic effect is only observable when both components are present in the organism.

    STZ, while this fraction only made up about 30% in urine from pigs. This means that although the elimination rate ( t , p and CIB) was approximately the same in the two species, the mechanisms of elimination were quite different. Urinary excretion contributed most to elimination of STZ from goats, while metabolism was the most important factor in pigs.

    Metabolism of STZ in goats involves two important pathways, N4-acetylation and oxidation, as does the metabolism of sulfadi- midine (Nielsen, 1973b) and sulfadiazine (Atef & Nielsen, 1975). Although "1- acetylation of sulfonamides is an important metabolic pathway, it seems to play a less important role in goats than in other animal species. In pigs, N'-acetylation of STZ is by far the most important metabolic pathway, and as much as 70% of the sulfonamide excreted in urine consisted of N4-acetyl-STZ.

    As STZ and some other sulfonamides are currently used in combination with trimethoprim, it is of interest to compare the half-lives of these sulfonamides with that of trimethoprim in domestic animals (Table 11). Sulfadiazine and sulfamethoxazole are elimin- ated from plasma of pigs, goats and cows a...


View more >