Drug disposition and biotransformation in the developing beagle dog

29-37(1988)

FUNDAMENTALANDAPPLIEDTOXlCOLOGY1~,

Drug Disposition

and Biotransformation

D. J. ECOBICHON,*,’ *Department

in the Developing

A. S. D’VER,?

AND W. EHRHART~

ofPharmacology and Therapeutics, McGill University Mont&al, and t White Eagle Laboratories, Doylestown, Pennsylvania

Received

April

9, 1987: accepted

Beagle Dog’

January

Quebec, 18901

Canada

H3G

1 Y6.

l&I988

Drug Disposition and Biotransformation in the Developing Beagle Dog. ECOBICHON, D. J., A. S., AND EHRHART, W. (1988). Fundam. Appl. Toxicol. 11, 29-37. The ability of developing male and female beagle pups to biotransform and eliminate drugs was studied by administering single intravenous doses of acetaminophen (50 mg/kg body wt), phenobarbital (15 mg/kg body wt), or phenytoin (15 mg/kg body wt) to the same groups of dogs (n = 6-8/ drug) at 4. 10, 20, 40, and 60 days of age. At suitable intervals after treatment, small (1 .O ml) blood samples were obtained via the jugular vein and centrifuged and the plasma was recovered and stored at -20°C to await analysis. Acetaminophen proved to be the most interesting “probe” of function with the plasma elimination half-life @t/2) in 40- to 60-day-old pups being 4.5-fold shorter than at 4 days of age. The synthesis of sulfate-conjugated drug decreased with age. In older pups, the synthesis of the glucuronide-conjugated drug was predominant. The elimination of sulfated acetaminophen from plasma was slow at all ages whereas the rate of glucuronide disappearance increased with age. Phenobarbital was slowly eliminated from the plasma at all ages and there was no indication of phydroxylated metabolite formation. The plasma Pt/2 of phenytoin decreased dramatically with age, a IO-fold difference occurring between 4- and 60day-old pups. para-Hydroxylated phenytoin (pHPPH) was detected only in the plasma of 4- and IO-day-old pups, the plasma Pt/2 decreasing with age. With the appropriate chemical and using the technique of collecting small, serial blood samples, this animal model can be potentially useful in perinatal toxicity studies. 0 1988 Society ofToxicology. D'VER,

The potential exposure of the developing fetal and neonatal human to a variety of xenobiotic agents has resulted in considerable interest in the study of physiological and biochemical mechanisms of elimination and detoxification of acquired foreign chemicals (Horning et al., 1975; Neubert et al., 1978: Juchau et al., 1980; Schwarz and Yaffe, 1980; Aranda and Stern, 1983). Surrogate animal models have been of limited use in describing what occurs in the perinatal human because

of the immaturity of tissue function of most species during gestation, with the possible exception of the perinatal guinea pig and lamb (Short et al., 1976; Lui et al., 1980; Pak and Ecobichon, 1982; Brien et al., 1985; Szeto, 1982). Interesting studies of caffeine biotransformation have been conducted in newborn dogs, the plasma elimination half-life and rates and routes of biotransformation being similar to those measured in the newborn human (Warszawski et al., 1977; Aldridge and Neims, 1980). In selecting the perinatal dog as a potentially useful model, we wanted to use the same animals at different ages, using the least invasive technique possible to examine the postnatal development of drug detoxifying enzymatic systems by collecting small

’ This research was presented as a poster at the International Congress on Toxicology, Tokyo, Japan, July 2 l25. 1986 (Toxicol. Left Sl(Supp1. I), Abstract Pl2-6, 1986). ‘To whom correspondence and reprint requests should be addressed. 29

0272-0590188 $3.00 Copyright 0 1988 by the Society ofToxicology. All rights of reproduction in any form reserved.

30

ECOBICHON,

D’VER, AND EHRHART

serial blood samples following treatment by intravenous injection. As “probes” of detoxification pathways, three commonly used drugs, acetaminophen, phenytoin, and phenobarbital were selected, their biotransformation being well known. Acetaminophen was chosen since, at moderate doses, it undergoes PHASE II (glucuronidation, sulfation) detoxification without activation by tissue cytochrome P-450 monooxygenases (Mitchell et al., 1974; Hinson et al., 1981; Wong et al., 198 1). Phenytoin is converted by PHASE I monooxygenases to a hydroxylated intermediate prior to PHASE II detoxification while phenobarbital is only slowly oxidized to a hydroxylated derivative (Gerber et al., 1972; Whyte and Dekaban, 1977). The objectives of this investigation were to study the disposition of the three drugs in the blood, measuring (1) the rate of disappearance of the administered drug; (2) the appearance and quantity of primary metabolites or detoxification products; and (3) the rate of disappearance of these products following the administration of single, intravenous, “challenge” doses of these agents to small groups of male and female beagle pups at selected intervals between 4 and 60 days after birth. MATERIALS

AND

METHODS

Chemicals. Sodium phenobarbital was purchased from Allen and Hanburys, Toronto, Ontario, while phenytoin and acetaminophen were purchased from Sigma Chemical Co., St. Louis, Missouri. Solutions for intravenous injection were prepared in sterile physiological saline, alkalinizing the solutions with sufficient 1.O M sodium hydroxide to dissolve the phenytoin and acetaminophen. Animals. Healthy purebred male and female beagle pups (White Eagle Laboratories, Doylestown, PA) were selected at 2 days of age and were assigned in a random fashion between three groups containing 6-8 animals/ group (3-4 of each sex/group). The pups were housed with their dams between experiments and were allowed to nurse freely. No special treatment of the pups was necessary beyond the routine vaccination, etc. Treatment. Single doses of acetaminophen (50 mg/kg body wt), phenytoin (15 mg/kg body wt) or sodium phenobarbital (15 mg/kg body wt) were administered intra-

venously in the jugular vein of pups at 4, 10, 20, 30 or 40, and 60 days of age using, wherever possible, the same group of dogs throughout the study with one drug. At suitable intervals after drug administration (0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hr), small blood samples (1.0 ml) were obtained via syringe and heparinized vacuum tubes from the jugular vein. The samples were centrifuged and the plasma was removed and stored at -20°C to await analysis. Analysis. Since only the unbound drug or metabolite could be assayedusing this microtechnique, it was essential to determine the extent of binding of the drugs to dog plasma over a wide range of concentrations in vitro. Pooled plasma from adult beagles was incubated for 10 min at 25°C with concentrations ofacetaminophen, phenytoin, or phenobarbital ranging from 2.5 to 50 mg/ml. Aliquots (0.4 ml) of the plasma were pipetted into Amicon Centrifree micropartition tubes (Amicon Canada Ltd., Oakville, Ontario) and centrifuged for 15 min at 3000g in a clinical centrifuge to separate the free (unbound) fraction of the drug into the protein-free ultrafiltrate. Similar experiments were carried out using pooled plasma from beagle pups of 2,5, 10, 20, 30,60, and 90 days of age, incubating aliquots with the drugs at concentrations of either 5.0 or 50.0 mg/ml followed by separating the free fraction of the drug by ultrafiltration. The ultrafiltrate of the plasma or a suitable dilution was injected directly on a high performance liquid chromatograph (HPLC) (Perkin Elmer, Series 10) equipped with a Shandon C Hypersil-ODS reverse phase column (25 X 0.46 cm) of 5.0-pm pore size (Chromatography Sciences Co., Inc., Montreal, Canada). For the analysis of acetaminophen and its biotransformation products. the mobile phase was water:methanol:acetic acid (86.5: 12.5: 1.O) (Hidvegi and Ecobichon, 1986). For the analysis of phenytoin, phenobarbital, and their primary hydroxylated metabolites, the mobile phase was a 40~60 mixture of methanol:NaHzP03 buffer, 0.025 M, pH 8.0 (Dykeman and Ecobichon, 1979). The column effluent was monitored at 0.02 absorbance full scale (aufs) at 254 nm, the absorbance change being recorded on a strip chart recorder. The quantitation of drugs and biotransformation products was done by comparing peak heights of unknown with those of known amounts of agent(s) interspersed between the analyses and used to prepare standard curves. Standard curves were constructed by plotting peak heights (mm) versus drug concentration (@g/ml). The relationships were linear over a concentration range of 0.2-50 pg/ml for phenytoin and phenobarbital and of 0.1-2.0 fig/ml for acetaminophen and the glucuronide and sulfate derivatives. The correlation coefficients for the calibration curves for phenytoin, phenobarbital, and acetaminophen were 0.997, 0.994, and 0.989, respectively, these values being derived from 10 individual curves for each agent. The detection limits for phenytoin, phenobarbital, and acetaminophen were 0.5, 0.25, and 0.2 @g/ml, respectively. The detection limits

DRUG DISPOSITION

10 ----

7-m 40

20

PLASMA

DRUG

60

( ug/ml)

FIG. 1. The recovery of “free” or unbound drug from the blood plasma ofbeagle dogs treated in vitro with varying concentrations of acetaminophen, phenobarbital, and phenytoin. Each point is the mean of three independent samples. See Methods section for details of experiment.

for the glucuronide and sulfate of acetaminophen were comparable to that for acetaminophen (Dykeman and Ecobichon. 1979: Hidvegi and Ecobichon. 1986).

RESULTS The results of in vitro binding studies with adult beagle plasma are shown in Fig. 1. Acetaminophen was 90% free over the entire concentration range tested. Phenobarbital was highly bound (60%) at low concentrations but, as the plasma drug concentration increased, a higher proportion existed as free drug. Phenytoin was extensively bound (7080%) over the entire range of concentrations tested, only 20-30% being in the unbound form. Experience with perinatal guinea pig plasma, in other studies, demonstrated that the fraction of bound drug varied considerably with the age of the animal, more unbound drug being available for distribution in younger animals (Pak and Ecobichon, 1982). Whether a similar phenomenon would be found in beagle pups was tested by treating aliquots of pooled blood plasma from pups ranging from 2 to 90 days of age with either a low (5.0 yg/ml) or a high (50 fig/ml) concentration of acetaminophen, phenytoin, or phenobarbital. The results are shown in Table 1. There was no change in the level of unbound acetaminophen (90%) in plasma at the vari-

IN PERINATAL

31

DOGS

ous stages studied. However, unbound phenobarbital decreased from 89% to 68.5% over the range from 2 to 90 days of age at the low concentration with a smaller decrease in unbound phenobarbital being observed over the same age range at the 50 bg/ml level. Phenytoin binding to pup plasma proteins was not as extensive as that measured in the adult beagles (Fig. 1). Free phenytoin decreased from a level of 54% at 2 days of age to a level of 28.5% at 90 days of age at the lower dose tested. At the higher dose, the amount of recoverable, unbound drug measured was 58.5% at the youngest age, decreasing to 36% by 90 days of age. Acetaminophen proved to be the most interesting “probe” of biological function, particularly in that it tested the ability of the animals to conjugate this agent with glucuronic acid and/or sulfate. In Fig. 2 are shown the time-blood plasma concentration relationships for the same pups at different ages. Similar initial plasma concentrations were seen at all ages and the rate of elimination of acetaminophen from the plasma increased with age until, at 40 or 60 days of age, there was

TABLE I INVITRO BINDINGOFPHENYTOINANDPHENOBARBITALTOPLASMAPROTEINSOFBEAGLEFTJPSOFDIFFTRENTAGES

Percentage unbound drug” Phenobarbital Days of age 2 5 10 20 30 60 90 Adult

5.0 &ml 89.0 87.5 81.5 87.5 75.0 75.0 68.5 50.0

50 *g/ml 91.0 88.5 92.5 99.0 88.5 88.5 82.5 80.0

Phenytoin 5.0 &ml

50 &ml

54.0 57.5 50.0 50.0 40.0 29.0 28.5 25.5

58.5 59.0 53.5 53.5 44.0 34.5 36.0 32.5

’ Each value is the mean percentage of unbound drug determined from three separate binding experiments at each age and dose level.

32

ECOBICHON,

ISVER. AND EHRHART

i

0.2 1 . 0

2

6

s TIME

8

1.0

12

( hr I

FIG. 2. The disappearance of “free” acetaminophen from the blood plasma of male and female (n = 8) beagle pups at different ages, demonstrating a more rapid removal of the drug as the animals matured. Each dog received 50 mg acetaminophen/kg body wt by intravenous injection. Each point shows the mean plasma concentration SD for eight individual animals.

essentially no difference. Figure 3 shows the formation and the rate of disappearance of acetaminophen glucuronide (left panel) and sulfate (right panel) from the plasma of the pups at different ages. Sulfate-conjugated drug, peak levels predominant in the plasma of pups at 4 days of age, decreased with age of the animals. Peak levels of sulfated acetaminophen were attained by 2 hr post-treatment in dogs of 4 and 10 days of age. In older dogs, lower peak levels of sulfate derivative occurred by OS- 1.O hr after treatment. The rate of disappearance of the sulfate conjugate from the blood plasma was comparable in pups of all ages. In the very young pups (410 days of age), glucuronide formation appeared to be slow, peak plasma levels being attained at 2-3 hr post-treatment. The rate of disappearance of glucuronide from the plasma was also slow in the very young dogs.

As the animals became older, the rate of glucuronidation became more rapid as was evidenced by the earlier appearance in the blood at slightly higher concentrations. The rate of glucuronide disappearance from the plasma increased with the increase in age, the 40- and 60-day-old pups being comparable. The rate of elimination of sodium phenobarbital from the blood plasma of beagle pups at different ages is shown in Fig. 4. The drug was slowly eliminated at all ages. The plasma distribution curve for the 4-day-old pups was markedly different from the curves for the intravenous injections into older dogs, the peak plasma concentration being attained only 4 hr post-treatment. The reason for this bizarre curve is not understood. The biotransformation of this drug appeared to be nonexistent in the beagle pup since, at no age interval, was any hydroxylated intermediate detected. The elimination of phenytoin from the blood plasma of beagle pups at different ages is shown in Fig. 5A. The low levels of drug detected reflect free or unbound drug. Phenytoin disappeared slowly from the blood stream of beagle dogs at 4 and 10 days of age but, by 20 days of age, there was little difference in the rate of disappearance from that observed in 30- or 60-day-old dogs. Small amounts of unbound hydroxyphenytoin were detected in the blood plasma of beagle pups at 4 and 10 days of age, the rate of elimination being somewhat more rapid in the 1Oday-old animals (Fig. 5B). No trace of this metabolite was detectable in the plasma of dogs 20 days of age or older, even in the earliest samples obtained. In Table 2 are shown the plasma half-life (@t/2) values for acetaminophen and phenytoin calculated from best fit lines from the data plotted for each of the pups at each age interval studied. As was reflected in Figs. 2 and 5, the Pt/2 decreased rapidly with age. Reliable estimates of the plasma half-life of phenobarbital could not be obtained from the assay data since samples were not collected over a long enough period of time for this slowly eliminated agent.

DRUG DISPOSITION

IN PERINATAL

33

DOGS

J. 0

2

4

5 TIME

5 (hrl

10

12

0

2

4

5 TIME

5

to

12

(hr I

FIG. 3. The presence and disappearance of acetaminophen glucuronide (left panel) and acetaminophensulfate (right panel) in the blood plasma of the male and female beagle pups (n = 8) treated with acetaminophen (50 mg/kg body wt) at different ages. Each point represents the mean plasma concentration SD for eight individual animals.

DISCUSSION

xenobiotics, these differences being particularly significant in the conjugation or PHASE II biotransformation mechanisms. Many speThe newborn infant is exposed to a variety cies conjugate drugs such as acetaminophen of chemicals not only throughout gestation with both glucuronic acid and sulfate, large but during the course of parturition as well as in the postnatal period as a consequence of interspecies differences being measured in the ratio of the two products (Savides et al., 1984; direct administration of drugs to the mother or infant or by passive acquisition via the Hjelle et al., 1985; Hidvegi and Ecobichon, have recognized for milk (Yaffe and Stern, 1976: Aranda and 1986). Pediatricians Stern, 1983). Since the fetus is no longer at- many years that mechanisms for glucuronitached to the maternal organism, it is depen- dation are limited in the newborn and premadent upon the capabilities of its own tissues to ture infant, the enzyme glucuronyltransferase being virtually absent in fetal life and detoxify and eliminate potentially hazardous present only at reduced levels at birth (Lathe agents. The mechanisms involved in chemical detoxification, in addition to being func- and Walker, 1958; Dutton, 1959; Burchell, tionally immature in the newborn, are fre- 1974; Yaffe and Stern, 1976). In contrast, sul(diethylstilbestrol, morquently quite different from those existing in fate conjugation phine) in the human fetal liver proceeded at the older child and/or the adult. An additional important feature to be considered in a rate comparable to that found in adults the newborn is that the day-to-day status of (Percy and Yaffe, 1964; Namkung et al., these detoxification mechanisms changes 1977). Many sulfated derivatives of endogerapidly (Aranda and Stern, 1983). nous and exogenous agents have been deMost mammalian species have several al- tected in the perinatal period, alternatives to ternative pathways for the detoxification of those conjugated products preferably pro-

34

ECOBICHON,

DYER.

I

0

4

8

16

24

TIME

32

40

48

(hr)

FIG. 4. The disappearance of “free” phenobarbital from the blood plasma of male and female (n pups at different ages. Each dog received 15 barbital/kg body wt by intravenous injection. shows the mean plasma concentration SD for ual animals.

= 6) beagle mg phenoEach point six individ-

duced at later stages. Sulfation has been described as one of the most primitive of biochemical reactions, being in existence in organisms before the arrival of gaseous oxygen in the Earth’s atmosphere and using either adenosine-5-phosphosulfate (APS) or 3-phosphoadenosine-5-phosphosulfate (PAPS) as the source of sulfate (Dodgson, 1977). The same pattern of low or nonexistent glucuronyltransferase but relatively high sulfotransferase activities has been observed in animals (Burchell and Dutton, 1975; Ecobichon et al., 1978). Acetaminophen, at nontoxic doses, would appear to be a useful “probe” of pathways of biotransformation in the perinatal beagle dog. The results demonstrated that the young beagle possessed PHASE II biotransformation capabilities very early in life, being able to both glucuronidate and sulfate acetaminophen efficiently. From the curves shown in Fig. 3, it would appear that the rate of elimination of the sulfate derivative did not change

AND EHRHART

with increasing age. As is reflected in the plasma levels, acetaminophen sulfate production was highest at 4 days after birth but had decreased fourfold by 60 days of age. In contrast, the efficiency of eliminating both the parent drug and its glucuronide from the plasma increased with age, there being little difference in rates after 40 days of age. While the improved, presumably renal, clearance interfered with the interpretation of the results, the ability to form acetaminophen glucuronide appeared to increase with age, higher concentrations being found in plasma earlier in time at 40-60 days of age although the peak levels detected at 0.5- 1.O hr could not be considered as significantly higher from those measured at 2 hr post-treatment in younger animals (Fig. 3). The overall conclusion of the results would suggest that, in dogs older than 60 days of age, the predominant urinary product of acetaminophen would be the glucuronide, the sulfate conjugation pathway occupying only a minor role. Savides et al. (1984) reported that approximately 75% of acetaminophen administered to adult dogs appeared in the urine as glucuronide, while only I7- 19% existed as the sulfate. Phenobarbital and phenytoin would appear to be poor drugs to use in the study of the development of biotransformation pathways in the perinatal dog. Phenytoin, while it proved to be a good chemical “probe” for biotransformation pathways, posed other significant problems because it was tightly bound to the plasma proteins and could not be efficiently recovered by the micropartition technique used. Phenytoin did undergo hydroxylation to 5-(p-hydroxyphenyl)-5-phenylhydantoin (pHPPH) in the beagle pup, this intermediate being detected in the plasma of animals at 4- 10 days of age (Fig. 5B) (Butler et al., 1977; Dykeman and Ecobichon, 1979). Since this hydroxylated derivative is known to be conjugated with glucuranic acid, the absence of the unconjugated intermediate in the blood of older dogs was not surprising if the intermediate was more efficiently glucuronidated and eliminated in

DRUG DISPOSITION

IN PERINATAL

35

DOGS

B

? 0

2

4

6

8

10

12-

TIMEthr)

TIME(hr)

FIG. 5. The disappearance of “free” phenytoin (A) and hydroxyphenytoin (B) from the blood plasma of male and female (n = 8) beagle pups at different ages. Each animal received 15 mg phenytoimkg body wt by intravenous injection. Each point shows the mean plasma concentration SD for eight individual animals. Hydroxyphenytoin was not detected in the plasma samples of dogs older than 10 days of age.

the older animals. The more efficient glucuronidation and elimination of acetaminophen by beagles of 20-60 days of age would

TABLE 2 PLASMA HALF-LIFE ESTIMATIONSFOR ACETAMINOPHENANDPHENYTOININBEAGLEPUPSOFDIFFERENT AGES P-phase plasma half-life” Days of age

Acetaminophen

4 10 20 30 40 60

108 f 14 min 84+ 8.1 min 36 f 3.3min 24? 2.0min 18? 2.0min

Phenytoin6 9.0 5.0 42 48 46

f 1.2 hr k 1.6 hr f 5.2 min +4.6min f 5.3 min

‘Values represent the mean half-life + SD derived from values obtained for individual plots of each treated pup (n = 6 or 8) at each time interval studied. b Acetaminophen and phenytoin were administered intravenously at dosages of 50 and I5 mg/kg body wt, respectively.

support this contention. The plasma elimination curves for phenobarbital were comparable at the different age intervals studied. Phenobarbital did not appear to undergo significant p-hydroxylation in the young dogs, no intermediate being measured at the detection limit of 1.5 pg/ml (Dykeman and Ecobichon, 1979). Certain hepatic, microsomal cytochrome P-450 monooxygenases are known to develop rapidly in the beagle pup after birth, Aldridge and Neims (1980) having shown a marked change in the N-demethylation of caffeine to theophylline/paraxanthine by 6-15 days of age. It is possible that the metabolism of phenobarbital was slow and that the low level ofp-hydroxyphenobarbital formed was rapidly glucuronidated and eliminated. However, this hypothesis was not supported by the plasma elimination curves for phenobarbital. The usefulness of the neonatal beagle dog as a surrogate model for the human infant must be considered in the light of the distinctive species differences in constitutive

36

ECOBICHON,

DYER,

PHASE I and II enzymatic activities in tissues as well as the substrate specificities for particular enzymes. In the present study, the status of the tissue microsomal monooxygenase activities of newborn pups cannot be clarified since, of phenobarbital and phenytoin, only the latter agent was converted to a detectable hydroxylated intermediate at an early age, suggestive of monooxygenase activities present in tissues. Other investigators have demonstrated a rapid postnatal development of tissue monooxygenases as is the situation with the human (Warszawski et al., 1977; Aldridge and Neims, 1980). At birth, the human infant is capable of synthesizing sulfate derivatives at rates comparable to those measured in adults while, without exception, reactions involving glucuronidation are quite low and only develop some months after birth (Dutton, 1959; Percy and Yaffe, 1964; Burchell, 1974; Namkung et al., 1977). In contrast, the beagle dog possesses relatively high and comparable levels of both sulfotransferases and glucuronyltransferases at birth. For the drug acetaminophen, the ability to form the sulfate conjugate decreases by 2 months of age and the predominant conjugated product becomes the glucuronide. ACKNOWLEDGMENTS This research was supported by the Medical Research Council ofcanada (Grant MA-36 11). The authors thank the staff of White Eagle Laboratories for their diligence and assistance in collecting blood samples.

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