Acta pharmacol. et toxicol. 1984, 54, 321-326.

From the Department of Pharmacology and Toxicology and 2, Chemistry Department. Royal Veterinary and Agricultural University, 13 Biilowsvej, DK-I 870 Copenhagen V, Denmark

Pharmacokinetics and Metabolism of Sulphadiazine in Neonatal and Young Pigs

BY Christian Friisl), Nils Gyrd-Hansen’), Pout Nielsen I), Carl-Erik Olsen2) and Folke Rasmussen’)

(Received January 3, 1984; Accepted January 19, 1984)

Abstract: The pharmacokinetics of sulphadiazine was studied in newborn, 1 week, and 8 weeks old piglets after intravenous administration of 60 mg/kg. Kinetic parameters were calculated using a two compartment open model. Steady state volume of distribution averaged 0.62, 0.56. and 0.48 l/kg at birth, 1 week, and 8 weeks, respectively. Elimination half-life decreased from 455 min. at birth to 322 min. at 1 week and 157 min. at 8 weeks leading to a rise in body clearance from 0.99 to 2.20 ml/min./kg during the same age period. Urinary excretion data indicated that the increase in body clearance reflects maturational changes in both renal function and metdbOliC capacity. Although renal clearance increased several times more than metabolic clearance, metabolism remained the main contributor to elimination of SDZ at all ages. Metabolism of SDZ involves two important pathways ~ acetylation and aromatic hydroxylation; the former being well developed at birth, while the latter increased markedly during the age period studied.

Key-wordy; Sulphadiazine - pharmacokinetics - metabolism - newborn piglets.

Sulphadiazine (SDZ) is used alone or in combina- tion with trimethoprim for treatment of various infections. In man (Andreasen et af. 1978) as well as in domestic animals (Nielsen & Rasmussen 1977; Luther 1978; Sigel et al. 1981; Brown et al. 1983) the kinetics of SDZ is well established. Like other sulphonamides this agent is eliminated partly by renal excretion and partly by bi- otransformation involving acetylation, oxidation, and glucuronic acid conjugation (Woolley & Sigel 1979).

However, results obtained in adults may be of limited relevance to newborn and young individu- als as they differ in a number of ways from adults: Lower plasma protein concentration, higher ex- tracellular fluid volume. and reduced hepatic and renal functions - all of which influence drug dispo- sition (Assael 1982). Despite the use of SDZ in paediatric patients (Aarbakke et a/. 1983), little

data is available on its disposition in the develop- ing organism.

Previous experiments have demonstrated the neonatal pig to be a convenient model for study- ing drug disposition in early life (Svendsen 1976). Hence, the present study was undertaken to exam- ine kinetics and metabolism of SDZ during post- natal development in this species.

Materials and Methods

Animals. Experiments were performed on seven new- born (1-3 days), six I-week-old (8 days) and six 8-week- old (60-75 days) female piglets. Newborn and I-week- old piglets got milk from the sows only. Eight-week-old pigs were weaned at 50 days of age and were fed with a commercial fodder mixture. Sucking animals remained with the sows until immediately before use and were returned 4 hours after dosing.

Experimental procedure. Sulphadiazine was prepared

322 CHRISTIAN FRIIS ET AL.

as an aqueous solution (0.6 g SDZ, 0.6 ml 5N NaOH and water ad 10 ml) and a dose of 60 mg/kg b.wt. was administered intravenously into the right ear vein. Fifteen to 17 blood samples (0.5-1 ml) were obtained from the bijugular trunk 3 min. to 24 hours after dosing as shown in fig. I . The samples were drawn through a cannula in the smaller piglets and through a polyethylene catheter placed in the truncus via the left ear vein in the larger ones. Plasma was separated from heparinized blood and stored at - 20" prior to analysis. In 17 animals urine was collected quantitatively through a Foley catheter during the first 4 hours after SDZ admin- istration and stored as plasma.

Substances. Sulphadiazine (2-sulphanilamidopyrimid- ine, SDZ) was purchased from Mecobenzon A/S, Copen- hagen, Denmark. N4-acetylsulphadiazine (N4-acetyl- SDZ) and 2-sulphanilamido-4-hydroxypyrimidine (4'- OH-SDZ) were synthesized as described previously (Atef & Nielsen 1975).

Analytical methods. Quaniitation. The concentration of SDZ in plasma and urine was determined speetropho- tomctrically according to the method of Bratton & Mar- shall (1939). Total sulphonamide (SDZ + N4-acetyl- SDZ) was determined in the same samples after acid hydrolysis and the eonccntration of N4-acetyl-SDZ cal- culated as the difference between the Concentration be- fore and after hydrolysis.

The relative amounts of urinary metabolites with free amino groups were determined by thin-layer chromatog- raphy (TLC) as described by Nielsen (1973) with ethyl aeetate/mcthanol/water/acetic acid (955: 1:0.5, by vol.) as solvent.

Identification. The urinary metabolites of SDZ were isolated rrom TLC-plates (0.75 mm, silica gel G F 254, Merek) in two different solvent systems: ethyl acetate/ methanol/water/acetic acid (955: I :0.5, by vol.) and chloroform/methanol (80:30 v/v). The two main meta- bolites were extracted from plates developed in the for- mer system by ethyl acetate and identified by means of mass spectrometry, HI-NMR spectroscopy and gas chromatography.

Electron-impact mass spectra were obtained on an AEI MS 30 instrument using direct inlet at 180-200". Ionization potential 70 eV, source temperature 200".

NMR spectra were run on a Bruker HX-90E instru- ment equipped with pulse/FT facilities. The samples were dissolved in 0.5-1 N deuterium ehloride/deuterium oxide. Sodium trimethylsilyltetradeuteriopropionate was used as internal standard.

Gas liquid chromatography was performed on a PYE- UNICAM GCV gas chromatograph equipped with a flame ionization detector (Column 1.6 m, 2 mm i.d.; stationary phase, 3% OV-101 on 80/100-mesh WHP; carrier, N2; injector temp., 280"; detector temp., 350"; column temp., programmed from 230" to 280" at S o / rnin.). Methyl/irimethylsilyl derivatives were madc by treatment of the samples with diazomethane in ether/ methanol 1:l for 5 min. at room temperature. After

evaporation the products were silylated with a mixturc of 5 drops of N,O-bis-trimethylsilyl-trifluoroacetamidc and 2 drops of dimethylformamide for 10 min. at 120'.

Analysis ofdata. The plasma SDZ data of each animal were analysed by a nonlinear iterative curve-fitting pro- gram AUTOAN (Sedman & Wagner 1976). Pharmacoki- netic parameters were calculated using standard equa- tions (Wagner 1976). The renal clearance (CIR) was cal- culated by dividing the urinary excretion rate of SDZ by the plasma concentration at the midpoint of the col- lection period. This concentration was computed from the non-linear fit. Metabolic clearance (C~M) was assu- med to be the difference between body clearance (CIB) and renal clearance.

Results

Pharmacokinetics. The plasma concentration of SDZ showed a biex- ponential decline and fitted a two-compartment open model in all three age groups (fig. 1). The kinetic parameters determined according to this model are summarized in table I . Distribution half-life (tliza) was short at an early postnatal age but tended to increase at the end of the period under study. The steady state volume of distribu- tion (Vdss) decreased slightly from 0.62 l/kg at birth to 0.48 I/kg at 8 weeks of age reflecting changes in both the central (Vi) and the peripheral compartment (V2). Maturation did not affect the distribution of SDZ between the body compart- ments since the ratio vI/vdss remained constant with age. Elimination half-life (tliZB) decreased markedly from 445 min. at birth to 322 min. at 1 week and 157 min. at 8 weeks leading to a rise in body clearance (CIB) from 0.99 to 2.20 ml/min./ kg during the same age period.

Renal (CIR) and metabolic clearance ( C ~ M ) of SDZ are shown in table 2. C1R remained constant at a level of 0.15 ml/min./kg during the first week of life but increased markedly thereafter to 0.8 1. ml/min./kg at 8 weeks. Thus CIR averaged 14% of body clearance in the younger piglets compared to 37% in the oldest ones. ClM increased gradually from 0.89 ml/min./kg at birth to 1.38 ml/min./kg at 8 weeks corresponding to 86% and 63% of the body clearance respectively.

Ident$cation of metabolites. TLC performed on urine samples from 8 weeks

PHARMACOKINETICS OF SULPHADIAZINE IN PIGLETS 323

200 400 600 800 min 1 100

50

20

10

200 r 7 - s

200 400 600 800 min

. . 0 200 400 600 800 rnin

Fig. I . Plasma concentration of SDZ following intrave- nous administration of 60 mg/kg b.wt. to three piglets aged 1, 8, and 60 days, respectively.

old piglets revealed the presence of unchanged SDZ, two major metabolites (N4-acetyl-SDZ and 4'-OH-SDZ) and a minor fraction of conjugated derivatives. Identification of the two major metab- olites were based on the following observations:

fl-acetyl-SDZ. This compound did not react with p-dimethyl-amino-benzaldehyde suggesting a substituted N4-amino group, and its chromato- graphic and mass spectral data were identical to those of reference material. The mass spectrum revealed a base peak at mjz 228 [M-S02]+ and intense ions at mjz 227, 186, 185, 140, 134. 125, 108, 95, and 92.

4'-OH-SDZ. This metabolite reacted with p- dimethyl-amino-benzaldehyde indicating a free N4-amino-group. The mass spectrum revealed ions at mjz 266 M', 202 [M-SOz]' 160, 156, 140, 11 1 , 108, and 92, indicating the presence of one additional oxygen atom in the SDZ molecule. The NMR spectrum showed a pattern typical for the para-disubstituted benzene ring with doublets at 8.13 and 7.66 ppm. The proton signals for the pyrimidine ring were markedly altered compared to that of SDZ, showing a multiplet superimposed on the high field signals from the benzene ring. This suggests a 4-hydroxy-pyrimidine ring rather than a 5-hydroxy-pyrimidine ring, which would give a singlet. The GLC retention time was found to be 5.96 min. for the methyl/trimethylsilyl de-

Table I . Pharmacokinetic data determined after intravenous administration of sulphadiazine (60 mg/kg) to newborn (1-3 days), 1 week-old (8 days), and 8-week-old piglets (60-75 days). Age 1-3 days 8 days 60-75 days

n 7 6 6 b.wt., kg 1.7 5 0.4 2 .7k l.0t l8 .6k 3.4t* A#glmll 47.0k37.8 43.0 k 27.9 55.1 k 7.6 a,min.- 0.178 k0.094 0.292 + O . I49 0.098 k0.034*

p,min:-' 0.001 59 k 0.00024 0.00221 + 0.00041 t 0.00445 k0.00045t* 5.0k2.7 3.0k 1.5 7.7 k 2.3*

445 k 62 322 & 54t 157&16t* 0.34+ O.02t di, (central) I/kg 0.44k0.09 0.41 k0.08

Vz, (peripheral) l/kg 0.18 k0.09 0. IS k0.08 0.14k0.03 Vdsr, l/kg 0.62+0.03 0.56 k 0.07 0.48 k0.04t* CIB, ml/min./kg 0.99 k0.12 1.23 +0.19t 2.20+0.337*

Mean k S.D. t significantly different from piglets aged 1-3 days, P<0.05. * significantly different from piglets aged 8 days, P<0.05.

-

B>Pg/ml 95.7k4.8 108.0k 14.4 120 .0~11.1

t ,120.m:n. t, ,p.mln.

324 CHRISTIAN P K l l S C l A L .

Table 2. Renal clearance (CIR), metabolic clearance (CIM) and body clearance (CIS) of newborn (1-3 days), I-week-old (8 days) and 8-week-old piglets (60-75 days).

~ ~ ~ ~ ~ ~ ___ Age 1-3 days 8 days ~ 60-75 d a y s

n 5 6 6 Urine pH 5.9k0.8 5.4k0.4 5.9 k 0.4 CIR ml/min./kg 0.15&0.15 0.16 0.18 0.81 &0.37t* CIM ml/min./kg 0.89 0.13 1.07 +0.13t 1.38 +0.08t* Clu ml/min./kg 1.04+ 0.09 1.23_+0.19 2.20 *

mcankS.D. t significantly different from piglets aged 1-3 days, P < 0.05 * significantly different from piglets aged 8 days, P < 0.05.

nvative compared to 5.17 min. for the correspond- ing unsubstituted SDZ derivative. Identical chro- matographic and spectral characteristics was shown for a synthetic sample of 4l-OH-SDZ.

#-acetyl-SDZ in plasma. The concentration of the main metabolite in plasma, N4-acetyl-SDZ, was determined between 3 and 4 hours after dosing. N4-acetyl-SDZ avera- ged 22 f 3, 28 & 5, and 22 4% of total drug in newborn, 1-week and 8-week-old animals

30-yo of dose

20-

(5) (6)

x

- 7 5 days

Fig. 2. Urinary excretion (0-4 hrs) of SDZ H, N4-acetyl- SDZ 0, 4'-OH-SDZ Elq and conjugated metabolites following intravenous administration of 60 mg/kg to newborn (1-3 days), I-week-old (8 days), and 8-week- old piglets (60-75 days) (mean+S.D.). The number of animals in each group is shown in the brackets. * significantly different from newborn and I week old animals.

respectively, with no significant difference (P > 0.05) between the groups.

Urinary excretion of metabolites, Total urinary recovery of SDZ and its metabolites within 4 hours after dosing was 12 & 6% of the dose at birth, 16 _+ 9% at 1 week, and 48 _+ 8% at 8 weeks of age. Thus, total recovery remained nearly constant in the first week but increased markedly thereafter. A similar development was observed for each component although the rate of increase was more pronounced for unchanged SDZ and 4'-OH-SDZ than for N4-acetyl-SDZ and conjugated metabolites (fig. 2). In newborn and I-week-old pigs the N4-acetyl-SDZ was the predominant compound in urine accounting for 65-69% of the total amount excreted while 4'- OH-SDZ comprised less than 6%. In contrast, the fractions of N4-acetyl-SDZ and 4'-OH-SDZ were almost equal in the 8-week-old animals and ac- counted for 28% and 24% of the total drug, re- spectively.

Discussion

SDZ was rapidly distributed in the body although the distribution half-life (tli2J tended to increase with age. This trend might well reflect the age- related changes in tissue distribution observed by Nielsen et a/. (1982). In the first week of life the steady state volume of distribution (Vdss) exceeded the value found at 8 weeks which is in agreement with findings for other sulphonamides (Svendsen 1976; Righter et al. 1979). The difference in V ~ S S may be attributed to the age-related rise in plasma

PHARMACOKINETICS OF SULPHADIAZINE IN PIGLETS 325

protein binding of SDZ (Nielsen et al., unpub- lished results) as well as to a decrease in ex- tracellular fluid volume (Setiabudi et al. 1975). The Vdss in 8-week-old piglets equaled the values in adult pigs (Luther 1978).

The elimination half-life of SDZ (t1,2P) was about 3 times longer during the first days of life than at 8 weeks at which time adult level was reached (Luther 1978). However, the significance of this difference as an index of altered elimination rate with age is not clear, since tl,2P is a hybrid parameter affected by changes in both distribution and elimination (Perrier & Gibaldi 1974). The change in Vdss of SDZ with age indicates that body clearance (CIS) is a better index for compari- son of elimination in newborn and young animals.

Body clearance increased by a factor 1.2 in the first week of life and by a factor 1.8 over the subsequent 7 weeks reflecting maturational chan- ges in both renal function and metabolic capacity. Further insight into the elimination of SDZ in the postnatal period can be gained by examining the separate contribution of renal (CIR) and metabolic clearance (CIM).

Renal clearance of SDZ constituted only 14% of body clearance during the first week but increa- sed more than fivefold thereafter accounting for 37% of body clearance at 8 weeks (table 2). This rise occurred without any change in urine pH. Since renal handling of SDZ in the pig kidney in addition to glomerular filtration also includes tubular secretion and reabsorption (Friis, unpub- lished results) the age-related difference in renal clearance may be attributed to changes in all these functions. In piglets, glomerular filtration rate and tubular secretory capacity increase twofold and fourfold, respectively, between birth and 8 weeks of age (Friis 1979 & 1982) whereas the fraction of drug reabsorbed appears to decrease with age, due to changes in permeability (Friis 1981 & 1982). Thus, the low renal clearance of SDZ in the first week of life possibly reflects low filtration and secretion as well as high reabsorption.

Although metabolic clearance increased only slightly compared to renal clearance, metabolism remained the main contributor to elimination of SDZ during the entire age period studied (table 2 ) .

According to the urinary excretion data metab- olism of SDZ involves two important pathways, aromatic hydroxylation and N4-acetylation. The formation of 4'-OH-SDZ has not previously been demonstrated in pigs but has been reported to occur in considerable amounts in goats (Atef & Nielsen 1975) and to a small extent in rats (Wool- ley & Sigel 1979). Compared to 8-week-old ani- mals the newborn and 1-week-old piglets excreted extremely small quantities of 4'-OH-SDZ indicat- ing aromatic hydroxylation to be only slightly developed at birth. In vitro experiments using the 9000 x g supernatant fraction of pig liver homog- enates confirm these findings (Nielsen et al., un- published results).

In contrast, the acetylation pathway must be relatively well developed at birth since N4-acetyl- SDZ was the predominant compound in the urine of newborn and 1-week-old animals. This inter- pretation is further supported by the high amount of N4-acetyl-SDZ in plasma during the entire age period studied. Similar substantial acetylation during the first days postnatally has been demon- strated for sulphadimidine in pigs (Svendsen 1976) and calves (Nouws et al. 1983) and for sulphadox- ine in goats (Nielsen et al. 1978).

Acknowledgements This work was supported by the Danish Agri-

cultural and Veterinary Research Council (1 3- 1637).

The mass spectrometer was granted by the Danish Natural Science Research Council (13- 3101) and the Danish Agricultural and Veterinary Research Council (1 1-3686).

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