Metabolism of Trimethoprim in Neonatal and Young Pigs: Comparative in Vivo and in Vitro Studies

Acta pharrnacol. el ta.rico1. 1984, 55, 402409.

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

Metabolism of Trimethoprim in Neonatal and Young Pigs: Comparative in Vivo and in Vitro Studies

BY N. Gyrd-Hansen, C. Mi, P. Nielseo and Folke RIsmuesen

(Received June 20, 1984; Accepted July 23, 1984)

Abstract: Metabolism of trimethoprim (TMP) was investigated in in viva and in v i m experiments on 1 day (group A), 8 days (group B), and 60 days (group C) old piglets. In the in vivo studies piglets received an intravenous injection of 14C-trimethoprim. Urine was then collected for 3 hours after which the animals were killed. During the collection period 13,24, and 40% of the dose was excreted in the urine in group A, B, and C, respectively. Trimethoprim and the following metabolites: Metabolite 1 and 4, minor metabolites, and conjugates were determined in plasma, liver, kidney, urine, and bile. The results show that newborn piglets have little capacity for oxidation of TMP while the ability to conjugate with glucuronic acid and sulfate seems somewhat higher. During the following 8 weeks a marked increase in the oxidative as well as conjugative potential took place. The microsomal fractions of liver and kidney were used for the in vitra metabolism studies of TMP. No metabolic activity could be demonstrated in the kidney preparations. Oxidative demethylation was just detectable in livers from the newborn piglets but increased considerably with age. Glucuronidation of metabolite 4 took place in the liver preparations from all three groups but at the highest rate in group C. The development in metabolic capacity was found to be qualitatively similar in viva and in vitra.

Key-wordr: Trimethoprim - neonatal pigs - drug metabolism - tissue distribution - renal excretion - protein binding - in viva and in vitra studies.

Drug metabolism in animals can be investigated through in vivo and in vitro experiments. Such studies have shown newborn individuals to be immature with respect to metabolic capacity and therefore less capable of eliminating foreign compounds including drugs (Assael 1982). In pigs it takes, however, only approximately two months for the metabolic function to reach adult levels indicating a very rapid development of the enzyme systems involved (Short & Davis 1970; Short & Stith 1973; Svendsen 1976; Klinger 1982).

Recently a research project was taken up by this department involving investigation and com-

parison of drug metabolism under in vivo and in vitro conditions in 1-60 days old piglets. It is the purpose of the project to see whether similar developments can be observed in the metabolic activities of tissue homogenates as in intact animals (Friis el al. 1984a & b).

For the present study trimethoprim was chosen as test compound as apart from being eliminated by renal excretion it also undergoes biotransfor- mations involving oxidative demethylation and subsequent conjugation (glucuronidation and sulfation) of the major metabolites (Schwartz et al. 1970; Nielsen & Rasmussen 1975a).

METABOLISM OF TRIMETHOPRIM IN PIGLETS 403

Materials and Methods

Animals. Twentyfour female piglets were used for the present investigation - twelve for in vivo and twelve for in v i m experiments (table I). In both cases the pigs were divided into three groups according to age: Group A, B, and C as shown in the table.

In vivo experiments. The piglets received a dose of 5 mg/kg ( 5 pCi/kg) 14C-trimethoprim (14C-TMP) in a volume of one ml per kg b.wt. as an intravenous injection in an ear vein. Urine was collected quantitatively by means of a balloon catheter for 3 hours after which a blood sample was taken. The piglets were then sacrificed by decapitation and brain, heart, lungs, kidneys, and liver were removed and weighed. Samples of these tissues as well as of muscle and bile were collected for analysis and immediately cooled in ice. All samples were stored at -20".

In vitro experiments. The animals were sacrificed by decapitation. Kidneys and liver were excised and after removal of the gall bladder placed in icecold Tris/KCI buffer, pH 7.4 (0.25M Tris, 0.15M KCI). The tissues were blotted dry before being weighed whereupon samples were homogenized with 2 volumes of the Tris/KCI buffer using a Sorwall Omni-Mixer (DuPont Instru- ments) at 15,OOO r.p.m. for 2 x 10 sec. with the homo- genizer vessel surrounded by ice throughout the oper- ation. The homogenates were centrifuged at 9,000 x g for 30 min. at 4" and the resulting supernatant used as enzyme source for the oxidation reactions. An aliquot of the supernatant fluid was centrifuged at 100,000 x g for one hour at 4". The pellet was suspended in 0.45M Hepes buffer and used as enzyme source for the assay of glucuronide formation.

The oxidative reactions were measured in a medium containing the following constituents in a total volume of 6 ml20 mM phosphate buffer with pH 7.4: Nicotina- mide adenine dinucleotide phosphate, 4.3 pmol; glucose- 6-phosphate, 20 pmol; magnesium chloride 20 pmol; sodium chloride, 425 pmol; 9,000 x g supernatant fraction equivalent to 500 mg tissue and trimethoprim (TMP) 0.2 pmol or p-nitroanisole, 4.0 pmol as substrate (Beckett & Haya 1977).

The glucuronyltransferase activity was measured in a

medium containing uridine diphosphate glucuronic acid (UDPGA), 4.0 p o l ; 100,000 x g pellets equivalent to 667 mg tissue in 3 ml 0.3M Hepes buffer, pH 7.4, with trimethoprim met. 4 (see fig. I), 0.27 pmol or occasion- ally phenolphthalein, 1 .O pnol as substrate.

Para-nitroanisole and phenolphthalein were used as control substances in order to check the quality of the tissue preparations used.

Analytical methods. The total concentration of I4C- TMP and its metabolites (fig. 1) in body fluids and tissues was measured by liquid scintillation counting. Plasma and urine (100 pl) were counted in 3 ml scintillation liquid (Ready-Solv." HP, Beckman). Tissues and bile were oxidized by incubation with 0.5 N quaternary ammonium hydroxide in toluene (Soluene" 350, Pack- ard). After complete dissolution 8 ml water and 10 ml scintillation liquid (Dimilume" 30, Packard) were added and the samples counted. Standards were run along with the samples of body fluids and tissues.

The relative concentrations of unchanged TMP and of its metabolites were determined after separation by TLC using a mixture of chloroform - isopropanol - 25% aqueous ammonia (80:20:1 by vol.) for development of the plates. The bands were visualized under UV-light and the amounts of I4C quantified by liquid scintillation counting of the plate material. Metabolite 1 and 4 were not well separated by this method and were therefore measured together. The identity of TMP and its metabolites was confirmed by running the pure compounds along with the samples on the plates.

Urine samples were diluted 1 : lO with water and ad- justed to pH 4.5 before incubation with p-glucuronidase/ arylsulfatase (SUC d'helix pomatia). The reason for this dilution is to overcome inhibition of p-glucuronidase due to high concentrations of the substrate or to the presence of a competitive inhibitor probably D-glucaro- 1,44actone (Ho & Ho 1980). During the incubation the glucuronides are cleaved while sulfates are left unchanged (Nielsen & Dalgaard 1978). Incubated and non-incubated urine samples were chromatographed and the decrease in conjugates, which corresponded well to the increase in metabolite 1 and 4, was taken as the amount of glucuronides in the samples.

The concentration of unchanged TMP in plasma and

Table I . Age and body weight of piglets.

Group A Group B Group C"

In vivo Number 4 4 4 Age (days) 1-2 7-9 55-66 Body weight (kg) 1.6*0.2* 3.8k0.7 16*2

~~~

In vitro Number 4 4 4 Age (days) 1 7-9 5 4 6 7 Body weight (kg) 1.7k0.2 2.8k0.4 15+4

* mean+S.D. weaned at 50 days of age.

404 N. GYRD-HANSEN ET AL.

Table 2. Relative organ weights in 12 piglets (YO of b.wt.).

Group A Group B Group C

Brain 2.14f0.41* 1.07f0.23' 0.43f0.071.b Heart 0.74f0.07 0.70k0.06 0.48f0.01a.b Lung 1.68 f 0.18 1.44 f 0.07a I .26 k 0.16' Kidney 0.88k0.15 0.73k0.04 0.48f0.04hb Liver 2.59k0.18 2.60*0.11 2.39k0.24

meanfS.D. a significantly different from A

significantly different from B

Results In vivo experiments. The mean relative organ weights for the three groups of piglets are given in table 2 from which it is seen that apart from the liver all relative organ weights decrease with age.

Three hours after administration I4C-TMP (unchanged TMP and its metabolites) was present in the tissues in concentrations which for kidney, liver and lung were higher and for brain lower than in plasma (table 3). In heart and muscle the concentration of I4C-TMP equalled that in plasma. For kidney, liver, and lung the tissue- plasma ratio was lowest in the newborn pigs.

Protein binding Of TMP decreased with age being 75 f 2, 45 f 12, and 43 f 10% in group A,

plasma ultrafiltrates from in vivo experiments and in samples from in v i m experiments was determined by means of HPLC (Nordholm & Dalgaard 1982). Another HPLC method was used to measure the concentration of metabolite 4 in samples from the in vitro experiments (Nordholm & Dalgaard 1984).

The rate of o-demethylation of p-nitroanisole to p- nitrophenol was determined by measuring the nitrophe- no1 formed by the method of Netter (1960) as modified by Short & Davis (1970). The formation of phenolphthalein glucuronide was determined as described by Short & Davis (1970).

Protein binding of TMP was determined by equili- brium dialysis using a Dianorm apparatus immersed in 37" water for 90 min. The samples were dialyzed against a phosphate buffer, pH 7.38 (Ehrnebo e f al. 1971) throu'gh a Visking tubing membrane with an average pore size of 2.4 nm allowing molecules with MW less than 12,00&14,000 to pass.

For statistical calculations a significance level of P<O.O5 was used.

B, and C, respectively. The plasma concentrations of TMP at which protein binding was measured were 5.1fO.8, 1.8*0.4, and 1.1fO.3 pg/ml for the three groups, respectively.

The relative amounts of (1) TMP, (2) metabolite 1 and 4, (3) "minor metabolites", and (4) conjugates (fig. 1) were determined in plasma, liver, and kidney. The concentration of TMP itself was seen to decrease with age in both plasma, liver, and kidney (fig. 2, table 4). Metabolite 1 and 4 were found in relatively small amounts in plasma and kidney and in the livers of the younger pigs. An age-dependent increase in the proportion of these metabolites was seen in the liver only. Pigs from

M I N O R M E T A B O L I T E S M A I N L Y C O N J U G A T E D .

Fig. I . Structure of trimethoprim and some of its metabolites numbered according to Schwartz et al. (1970).


Table 3. Tissue distribution of 14C-trimethoprim in piglets (meanf S.D.).

Ratio: Tissue/Plasma

Group A B C A B C

Plasma 5.1 k0.6 2.4f0.4‘ 2.0 f0.4a - - - Brain 1.9k0.3 1.6f0.2 1.0+0.4a.b 0.4k0.1 0.7f 0.1 0.5 k 0. I b Heart 4.7 ;t 0.5 3.250.5’ 2.3 f0.6sib 0.9 f 0.1 1.3fO.la 1 . 1 f O . 1 P . b

Lung 9.7 k 1 .o 6.8 f 1.6’ 6.3 f 1.5’ 2.0 i 0 . 4 2.8 f 0.4‘ 3.2 k 0.7’ Kidney 1 I .3k 1.2 15.5f3.Ia 14.1 f2.3’ 2.3k0.5 6 .4 f0 .9 6.9f0.5‘ Liver 7.1k0.7 7.8 ;t 0.9 5.8+0.9.b 1.4k0.3 3.2 f 0 2 2.9f0.4O Muscle 4.1k0.4 3.0fOSa 1.8k0.5e.b 0.8f0.1 1.2* 0.1’ 0.9 + O . l b a significantly different from A

significantly different from B.

group C had more “minor metabolites” in their plasma and liver than pigs from group A. Conju- gates of metabolite 1 and 4 were found in much higher concentrations in plasma from the oldest piglets than from the younger ones. Also livers from group B and C contained more conjugates than those from group A. In the kidney there was

100 :I 30

10 L 1 2 3 4

1

LIVER

I 2 3 4 1

PLASMA

C 1

1 2 3 4 1 A 2 3 4 1

2 3 4

C

2 3 4

a steady increase of conjugates with age (fig. 2). During the 3-hour collection period approxi-

mately 13% of the 14C-TMP administered was excreted in urine by the one day old piglets while the animals in group C excreted about 40% of the dose during the same period of time (fig. 3). In the urine collected, pH was found to be 5.6 f0.4,

1

3 i A

& 1 2 3 4

KIDNEY

1- 2 3 4 1

C

2 3 4

Fig. 2. Distribution of trimethoprim and its metabolites in plasma, liver, and kidney from 1 day (A), 8 days (B), and 60 days (C) old piglets. 1 . Trimethoprim 2. Metabolite 1 and 4 3. Minor metabolites 4. Conjugates of metabolite 1 and 4. Ordinate: Percentage of total drug concentration. The bars indicate 1 S.D. 0 Significantly different from group A 0 Significantly different from group B.


URINE 57

A B C Fig. 3. Excretion of 14C-trimethoprim in urine from 1 day (A), 8 days (B), and 60 days (C) old piglets. Ordinate: Percentage of dose. The bars indicate 1 S.D. 0 Significantly different from group A 0 Significantly different from group B.

5.3k0.4, and 6.2k0.7 in group A, B, and C, respectively. Almost 80% of the radioactivity in urine from newborn piglets was due to TMP with 18% present as conjugate (fig. 4, table 4). The proportion of TMP in urine dropped with age while the relative amounts of conjugates - both glucuronides and sulfates - increased, and in group C made up nearly 75% of the I4C-TMP in urine. The ratios between glucuronides and sulfa-

10

3 I 1

A

JL 1 2 3 4

URINE

1 2 3 4 1 2 3

tes were 0.4, 1.2, and 2.4 in group A, B, and C, respectively. Unconjugated metabolite 1 and 4 and “minor metabolites” were present in urine, but only in small amounts.

In bile the radioactivity represented both unchanged drug, metabolite 1 and 4, “minor metabolites”, and conjugates (fig. 4, table 4). As in urine an age-dependent decrease in TMP and rise in conjugates were observed. However, in bile from newborn piglets “minor metabolites” ac- counted for as much as 23% of the radioactivity present. In the oldest piglets this proportion had dropped to 9%. Especially bile from group C contained low levels of metabolite 1 and 4.

In vitro experiments. Oxidative demethylation of TMP was just detectable in livers from newborn piglets (fig. 5A). The figure shows further that the activity of the involved enzyme system increased markedly during the first weeks of life. The activity of this enzyme system was, however, low compared to the one responsible for oxidative demethylation of p-nitroanisole. Metabolite 4 of TMP is conjugated with glucuronic acid and fig. 5B shows the results from experiments where the 100,000 x g microso-

BILE

1 2 3 4 1 2 3 4 1 2 3 4 I

Fig. 4. Distribution of trimethoprim and its metabolites in urine and bile from 1 day (A), 8 days (B), and 60 days (C) old piglets. 1. Trimethoprim 2. Metabolite 1 and 4 3. Minor metabolites 4. Conjugates of metabolite 1 and 4; hatched column: sulfates; open column: glucuronides. Ordinate: Percentage of total drug concentration. The bars indicate 1 S.D. 0 Significantly different from group A. 0 Significantly different from group B.


A TMP P-NITROANISOLE

'1

L1 0 10 20 JOmin

B TMP-MET4 PHENOLPHTHALEIN I

Fig. 5A. Oxidation of trimethoprim and p-nitroanisole incubated with 9,000 x g supernatant of liver homogenates. B. Glucuronidation of TMP-metabolite 4 and phenolphthalein incubated with the microsomal fraction of liver homogenates. 0 1 day old piglets; Abscissa: Incubation time (min.) Ordinate: Substrate transformed (pmol/g liver). The bars indicate 1 S.D.

8 days old piglets; A 60 days old piglets.

ma1 fraction of livers was incubated with metabolite 4. The activity of the involved enzyme system was higher at birth than for the oxidative system, but the increase in activity with age was smaller. In similar experiments with phenolphthalein as substrate the glucuronyl transferase activity was always found considerably higher (fig. 5B).

Neither oxidation of TMP nor glucuronidation of metabolite 4 could be demonstrated when kid-

ney tissue was used as enzyme source under identical experimental conditions.

Discussion

Plasma protein binding of TMP was found to decrease from 75 to 45% in piglets during the first week of life. This decrease is surprising since the total plasma protein concentration in piglets re- mains constant in the first two months after birth.

Table 4 Relative amounts of TMP and its metabolites in tissues and body fluids.

Per cent of total concentration (mean k S.D.) Tissue/ Fluid GrOuD TMP Metabolite I +4 Minor Metabolites Coniueates

A 9 5 5 1 1.0k0.2 1.0+0.1 2.8k0.7 Plasma B 73+5 2.5 + 2.4 4.2 f 2.9 2.1+3

C 55+4 1.5k0.6 3.2k1.2 4 0 k 4 ~~

A 91 + 2 1.1k0.3 3.4k1.1 4.2+ 1.9 Liver B 82+3 2.6k0.3 4.6+ 1.9 10+4

C 7 3 k 6 6.3k1.7 6.4 f 1.7 14+3

A 55+12 2.3k 1.2 2 3 k 6 Bile B 3 0 k 7 3.3k 1.1 13+5

C 15+6 1.0+0.3 9 + 4

l9+ 19 5 3 k 8 75L-9

A 90+2 1.6k0.6 3.0k0.4 6.0k 1.7 Kidney B 73+5 2.9k0.8 2.3k1.1 21 + 5

C 58+6 2.7+ 1.3 3.2 k0.8 36+5

A 7 8 k 4 2.0k0.6 1.3k0.7 13+ 1 5 + 3 Urine B 60+7 0.8k0.3 2.3k1.0 17+2 20+7

C 2 2 k 6 1.0k0.8 2.1 f 1.7 22+5 52*8

Sulphates Glucuronides


Furthermore the concentration of albumin - the protein fraction to which most drugs are bound - shows a marked increase during the same period (Svendsen et al. 1972). The decrease in protein binding of TMP seen in the young piglets is also inconsistent with the general rule, that protein binding of drugs drops with increasing concentrations as the content of unchanged TMP in plasma was higher in the newborn than in the 1 week old piglets (see table 3 and fig. 2).

The observed fall in protein binding of TMP may contribute to the simultaneous increase in the volume of distribution for TMP - from 0.78 to 1.32 I/kg b.wt. ~ seen in piglets during the first week of life (Friis et al. 1984b).

Tissue concentrations of I4C-TMP - with the exception of the brain - were either equal to or higher than the plasma concentrations, which is in correspondence with earlier findings in adult pigs (Nielsen & Rasmussen 1975b). For a number of organs the tissue-plasma ratio was higher in the older (group B and C) than in the newborn pigs (table 3), and thus in accordance with the distribution volume of TMP being higher - and above one I/kg - in 1 and 8 weeks old piglets than in newborn ones (Friis et al. 1984b). The higher tissue-plasma ratio in the older piglets may be explained by the observed decrease in plasma protein binding together with the postnatal reduction in extracellular fluid volume (Setiabudi et al. 1975).

TMP is eliminated partly by metabolism and partly by renal excretion (Nielsen & Rasmussen 197%). During the period studied there was a marked increase in the capacity of the liver to oxidize and conjugate TMP, which is reflected in the relative decrease of unchanged TMP and increase of conjugates seen in all samples (fig. 2 and 4). In urine the radioactivity was found predominantly as TMP and as conjugates, which consist of metabolite 1 and 4 as glucuronides and metabolite 4 as sulfate (Friis et al. 1984b). In the newborn piglets more sulfation than glucuronidation took place while the opposite was the case in the 8 weeks old pigs. However, this change in the ratio between glucuronidation and sulfation may to some extent be due to increased glucuronidation of metabolite I (which is not conjugated

with sulfate) as Friis et al. (1984a) could not show any clear increase in the glucuronide-sulfate ratio for metabolite 4.

The observations made in vivo correspond well to the results from the in vitro experiments, which showed a distinct increase in the hepatic ability to oxidize TMP and to conjugate metabolite 4 with glucuronic acid during the postnatal period (fig. 5). As oxidation of TMP is a prerequisite for conjugation and very little free metabolite 1 and 4 was found in the in vivo experiments it can be concluded that in piglets at all ages the capacity for conjugation is at least as high as for oxidation - the in vitro results indicate the same.

The metabolism of the reference compounds p- nitroanisole (oxidative demethylation) and phenolphthalein (glucuronidation) by the liver preparations was much faster than that of trimethoprim and metabolite 4 undergoing the same type of reactions. This points to the existence of differences in substrate affinities for the enzyme systems involved (Klinger 1982).

The metabolic rate for the reference compounds and its increase with age is in agreement with the observations made by Short & Davis (1970) in experiments with porcine liver preparations. However, in identical tests using kidney tissue negligible or no enzyme activity - as in the present study - could be found (Short & Davis 1970).

Besides metabolism renal excretion is of im- portance in the elimination of TMP. A pro- nounced postnatal increase in the proportion of the dose (I4C-TMP) excreted by the kidney - from 13 to 40% - was observed (fig. 3). One reason for this increase in excretion is the immaturity of the porcine kidney at birth, and the fact that mature functional level is reached within eight weeks (Friis 1979). However, as the percentage of unchanged TMP in urine during the first 8 weeks drops from 78 to 22%, the proportion of the dose eliminated by renal excretion (i.e. excreted as unchanged TMP) over the 3 hours collection period is nearly the same in all three age groups - around 10% (figs. 3 and 4). Consequently it is the considerable development in the capacity for metabolizing TMP that is the main contributor to the marked increase in body clearance for TMP,


from 1.2 to 11.8 ml/min./kg b.wt., observed in 1 to 60 days old piglets - and the corresponding decrease in elimination half-life from 485 to 120 min. (Friis et al. 1984a).

From the present investigations it can be concluded that oxidation and subsequent conjugation is the major metabolic pathway for TMP and that these processes - especially oxidation - are poorly developed in newborn piglets. During the first weeks of life there is a marked increase in the capacity of these two metabolic processes - especially in oxidation - which could be shown in vivo as well as in vitro. The correlation between in vivo and in vitro metabolism seems, however, to be mainly qualitative making it questionable to draw quantitative conclusions concerning a compound’s biotransformation from in vitro experiments.

Acknowledgements This study was supported by the Danish Agri-

cultural and Veterinary Research Council, grant no. 13-1637.

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Metabolism of Trimethoprim in Neonatal and Young Pigs: Comparative in Vivo and in Vitro Studies