Uptake of phenytoin by tissues of mongolian gerbils

Gen. Pharmac. Vol. 13, pp. 527 to 530, 1982

0306-3623/82/060527-04503.00/0 Copyright © 1982 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

UPTAKE OF PHENYTOIN BY TISSUES OF MONGOLIAN GERBILS A. NAVARRO-RuIz, G. LOPEZ-ORTEGA, S. SANDOVAL-ROJAS, R. VARGAS-RODRIGUEZ and P. GARZ6N Laboratorio de Bioquimica, Divisi6n de Biologia del Desarrollo, Unidad de Investigaci6nes BioM6dicas de Occidente, I.M.S.S. Guadalajara, Jal, Mexico (Received 27 April 1982) Abstract--1. Radioactive phenytoin was extracted from brain, cerebellum liver and kidney of i.p.

injected Mongolian gerbils that were killed at hourly intervals. 2. The drug was extracted by two procedures. Procedure I involved acidification and chloroform extraction, the other, procedure II, an alkaline extraction, acidification and solubilization in chloroform (II). 3. Procedures I and II yielded 98 and 93% recoveries, respectively, from experiments performed either in vivo or in vitro. 4. Concentrations of labeled phenytoin found in brain, cerebellum, liver and kidney from the first to the fifth hour were 7.7, 7.8, 5.2, 4.0 and 2.2% (I) and 10.6, 8.5, 7.2, 4.0 and 2.8% (I1) of the amount injected, correspondingly. 5. Concentrations of phenytoin in cerebellum were greater by a statistically significant margin than were those in other tissue at the third hour following the time of injection of radioactive phenytoin but liver and kidney concentrations were greater than cerebellum uptake at the fourth and fifth hour. 6. Brain showed the lowest incorporation of phenytoin throughout five hours.

INTRODUCTION Phenytoin continues to be the most i m p o r t a n t anticonvulsant drug used to control generalized seizures (Reynolds, 1975; Reynolds & Shorvon, 1981; J o h a n nessen & Henrikesen, 1980). However, m a n y side effects have been attributed to this drug (Gibson & Becker, 1968; H a r b i n s o n & Becker, 1969; Poswillo, 1974; De Vore & Woodbury, 1977), although none has been sufficiently i m p o r t a n t to preclude its use. Some are related to liver extensive necrosis associated with a n excess of inflammatory cells suggesting an immunologic involvement ( G h a t a k et al., 1976; Gallagher et al., 1973; Crawford & Jones, 1962; Floyd, 1961). O t h e r effects include the cerebellar loss of Purkinje cells, damage that frequently has been described in epileptic patients under prolonged treatment with p h e n y t o i n as well as in animals receiving varying dosages of the same drug (Hofmann, 1958; P u r o & Woodland, 1973; Del Cerro & Snider, 1967; Utterback, 1958; Haberland, 1962; Salcman et at., 1978). While information on morphological changes of cerebellum continues to accumulate, little information has been published regarding accumulation of phenytoin either n o r m a l or in a b n o r m a l tissue (Dam, 1970a,b; Nielsen et al., 1971; Ito, 1970). Therefore we decided to examine phenytoin uptake by the cerebellum of seizure-free M o n g o l i a n gerbils preliminary to performance of similar examinations in seizureinduced animals. EXPERIMENTAL PROCEDURES

A stock solution of labeled phenytoin were prepared by solubilization of diphenylhydantoin-5,5-(phenyl-l,4-3H) S.A. 47.5 Ci/mM in redistilled ethyl alcohol. Sufficient tracer was diluted in 0.85% sodium chloride 527

solution to yield a final concentration of 2.5/tCi/ml. Freshly redistilled acetone, diethyl-ether and chloroform as well as recently prepared 6 N HCI, 40% trichloroacetic acid and 1.0 N NaOH were used. Tissue homogenates were prepared by use of a Potter-Elvehjem homogenizer. They were centrifuged in a Sorval RC-5 centrifuge at 3000 g for 5 min. Pellets so obtained were digested with hyamine hydroxide and vortexed before solubilization in liquid scintillation counting solutions for radioactivity measurement. The latter were made with the aid of a Packard Tri-Carb spectrometer model 3390. Direct additions of radioactive phenytoin in vitro to homogenates of recently excised brain, cerebellum, liver and kidney were carried out in 9 mature Mongolian gerbils of both sexes. Subsequently, incubation with continuous shaking at 37°C for 10 min was followed by another homogenization and chloroform extraction. Radioactivity of the labeled drug was then measured in the extracts in liquid scintillation spectrometer. Tissue uptake of phenytoin was measured in vivo at hourly intervals during a 5 hr period. 45 adult Mongolian gerbils (U.I.B.O., I.M.S.S.-Guadalajara) of both sexes were injected i.p. with 2.5ttCi of diphenylhydantoin-5-5(phenyl-l,4-3H) (New England Nuclear) and were killed at five hourly intervals. Liver, kidney, sagitally divided brain and cerebellum were excised and rapidly immersed in 0.85~ sodium chloride solution, and maintained at 4°C. The tissue were wiped and weighed and then extracted by one of two phenytoin extraction procedures. Procedure I involved an initial hypotonic tissue lysis, then acidification and finally a phenytoin recovery in chloroform. Procedure II consisted in tissue homogenization with 1.0N NaOH, protein precipitation with acetone, and chloroform addition to the acetone-aqueous layer to eliminate acetone. Protein precipitation with 6 N HCI and a final chloroform extraction of the labeled phenytoin from the remaining aqueous layer were performed (Garz6n et al., 1981). Radioactivity measurement were then done. One way variance analysis and Student's t-test for paired samples were performed.

528

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RESULTS

The in vitro incubated tissue with labeled phenytoin showed 98 and 93% recoveries by procedures I and II, respectively. Negligible amounts of radioactivity were lost in aqueous layers, or in the pellets. Brain, cerebellum, liver and kidney showed uniform recoveries. These values support the use of either procedures I or II in terms of phenytoin recoveries. Figures 1 and 2 show that phenytoin recoveries are similar by both procedures. Also, total radioactivity showed to have been extracted both, from in vitro as well as from the in vivo drug exposed tissues. Table 1 presents radioactivities found in liver, brain, cerebellum and kidney following the i.p. inoculation of radioactive phenytoin. Mean drug uptakes of tissue during the first to the fifth hours were 7.7, 7.8,

5.4, 4.1 and 2.3~o by procedure I and 10.6. 8.5, 7.2, 4.0 and 2.8% by procedure II. As expected, liver accumulated more radioactivity when calculated in terms of total weight; however if expressed per gram of tissue. a more reliable and proportional comparison of tissue uptake was observed. Percentage of phenytoin recoveries per gram of each organs at each interval is showed in Fig. 3. Thus at the third hour, cerebellum accumulated 65% (P < 0.001) of the recovered radioactivity. Although its concentration had decreased at the fourth hour, it increased again to 47°0 (P < 0.001) at the fifth hour (I). The remaining tissues showed a marked increase in phenytoin content at the fourth hour, with a maximum value of 28% in liver and 26'I~; in kidney. No differences were detected for liver, kidney and brain tag content during the preceding three hours. A different pattern of drug uptake is shown in Fig. 4, where

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529

Table l. Percentage of radioactive phenytoin recovered from tissues* of Mongolian gerbils at hourly intervals 1

2

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4

5

7.7 ± 0.9 10.6 ± 0.7

7.8 ± 0.3 8.5 ± 0.4

5.2 ± 0.2 7.2 ± 0.6

4.0 ± 0.4 4.0 ± 0.3

2.2 ± 0.1 2.8 +_ 0.2

Procedures I

II

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Fig. 3. cerebellum is shown to accumulate more phenytoin per gram than the remaining tissues. Also at the third hour, a 48% (P < 0.001) recovery was observed. No differences in drug uptake were detected in liver and kidney at this interval. Brain represented only 8% of the total radioactivity (II). Figure 4 indicates that cerebellum accumulated less radioactivity on the fourth and fifth hours, decreased to 12 and 18% respectively, and corresponded to the amounts found in the brain at the same intervals. Liver and kidney radioactivity were found to be significantly elevated from the first to the fifth hours of the experimental periods (II). DISCUSSION

Phenytoin uptake by tissue has been measured previously by chemical and radiometrical methods (Noach et al., 1958; Dill et al., 1956). Maximum conPHENYTOIN

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centrations of phenytoin in all tissues as well as plasma were reported to occur 1.5 hr after its jugular injection. These authors also reported that liver accumulated more phenytoin than other tissues. Brain levels of the labeled drug were higher than those found in plasma of cats (Firemark et al., 1963) and corresponded to an initial tissue distribution phase of 2 hr in man (Gugler et al., 1967). Specific activity of the radioactive compound was modified with unlabeled phenytoin to obtain the desired doses per kg of body weight in their studies. This procedure was rejected in our experiments in order to avoid saturation of (possible) receptor sites with an excess of unlabeled drug. Also, because radioactive.phenytoin was wholly extracted directly (Garz6n et al., 1981) from tissue instead of being measured as accumulated radioactivity of digested tissues. It appeared that less interference would occur at the radioactivity measurement stage of procedures I and II and that more reliable comparisons would be possible. The effects of prolonged exposure to the drug were not considered in these studies in which we attempted only to measure tissue uptake of unmetabolized drug. No metabolites with radioactivity were detected by radiochromatography during a 5 hr incubation period. Measurement of tissue uptake of phenytoin revealed the initial 3 hr of the experimental period. Liver and kidney progressively accumulated more drug during the 4th and 5th hours which is to be expected if we consider their detoxifying role in the organism. Our findings support the possibility of'the existence of a connection between greater phenytoin uptake by cerebellum and production of damage by it reported to occur in this tissue in the form of cerebellar Purkinje cell disappearance following to prolonged exposure of epileptic patients to phenytoin (Puro & Woodward, 1973; Dam, 1970c,d, 1972; Snider, 1977; Rosichpla, 1980; Koller, 1980). Experiments performed in our laboratory have shown that phenytoin influences liver function and cellularoty (Garzdn et al., 1982).

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Acknowledgements The authors are indebted to Dr J. G. Reinhold for reviewing the manuscript and to Miss Judith Barba Borrego for typing it.

REFERENCES

lo0

Hours

Fig. 4.

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530

A. NAVARRO-RUIZ et al.

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