THE ELIMINATION OF HIGH DOSES OF PHENYTOIN

IN MAN AND THE RABBIT

by

Peter Chinwah

Department of Clinical Pharmacology

St. Vincent's Hospital

Darlinghurst, 2010, N.S.W.

AUSTRALIA

A Thesis submitted for the Degree of

Master of Science

in the

University of New South Wales

February, 1987 Ul·lilJ:2?.S1TY QF N.S.W. ~ 14 JUN 1988 f L!.:.": --":c_A:_'''"lARY I (i)

ACKNOWLEDGEMENTS

I wish to thank Professor Denis Wade for the opportunity to undertake this thesis in the Department of Clinical Pharmacology and for his overseeing and supervision of this project.

I would like to express my gratitude to Dr Garry Graham, Senior Lecturer in the Department of Physiology and Pharmacology at the University of New South Wales, for his significant contribution in discussion and critical review of this thesis.

I also wish to thank my colleagues of the Department of Clinical Pharmacology and Toxicology at St Vincent's Hospital for their assistance during the course of the project.

Finally, I am grateful to Dr Ken Williams of the Department of Clinical Pharmacology and Toxicology at St Vincent's Hospital for many hours of discussion, and his admonishment, long suffering and perserverence which have made this thesis possible. (ii)

ABSTRACT

The pharmacokinetics of phenytoin were studied in twelve patients who presented to casualty with phenytoin toxicity. These patients were divided into 3 groups according to the type of plasma elimination profiles observed.

Group 1. consisted of 3 patients who demonstrated first order elimination kinetics with long elimination half lives (70-106 hours). Group 2. consisted of 6 patients who showed saturable elimination kinetics (mean estimated terminal half life 28 hours). In Group 3. there were 3 patients who had "plateau" or rising plasma concentrations lasting from 2.5 - 14 days. It was thought that the rises and plateaus of plasma concentrations could be due to a) absorption and/or b) redistribution within body compartments.

The data demonstrates the large interpatient variability in the disposition of phenytoin following overdose. Clearly, the disposition of therapeutic doses of phenytoin cannot be used as a predictor of the time course of events following an overdose of the drug.

The factors leading to the plateau plasma concentrations were further investigated by studying the disposition of phenytoin in the rabbit. It was established that at a dosage of 74 mg/kg I.V. the plasma concentrations of phenytoin at first fell rapidly but then reached a plateau phase similar to that observed in man. During this plateau phase there was a significant relative change in phenytoin distribution (iii)

into fat when compared with a single therapeutic dose. There was 5.5% in fat following the low dose but 10-20% following the high dose. This data suggests that redistribution of phenytoin from fat back into plasma may be one of the factors contributing to the plateau of plasma concentrations seen in some patients following an overdose.

The protein binding of phenytoin was studied in the patients who had taken an overdose of the drug. Free fractions of phenytoin when first admitted and when plasma concentrations had returned to non-toxic concentrations were not significantly different. In contrast, however, there was a higher free fraction of phenytoin in plasma taken from rabbits given high doses of phenytoin as compared with those given a low dose. In this respect, the concentration dependent changes in protein binding in rabbits may contribute to the profiles observed, more so than in humans.

Transient hypotension has been reported in man after both I.V. and oral administration of phenytoin. An overall hypotensive effect of large doses of phenytoin was observed in rabbits. However, these changes in blood pressure were not sufficient to have an effect on the clearance of phenytoin. Liver blood flow was not decreased by large doses of phenytoin. It is unlikely that hypotensive effects of phenytoin significantly alter its disposition following overdose in man.

Large doses of phenytoin may not always result in toxic plasma concentrations. Some patients were found to require large doses of phenytoin for adequate control of their epilepsy. Short half (iv)

lives in these patients may be attributed to the effects of enzyme induction by other drugs taken concurrently. However, some people may be rapid metabolizers of phenytoin, although this could not be unequivocally demonstrated in these patients.

Patients who cleared phenytc:n rapidly did not necessarily clear other anticonvulsants rapidly. Epileptics with rapid metabolism of phenytoin may be better controlled by using a different anticonvulsant such as carbamazepine. (v)

PREFACE

Much of the work of this thesis has been published and/or presented at meetings of the Australasian Society of Clinical and Experimental Pharmacologists. During the course of the thesis, was involved in other projects which resulted in a number of publications which are also listed below:

Publications Supporting this Thesis

(1) Phenytoin, phenobarbitone and carbamazepine elimination kinetics in overdosed patients. P.M. Chinwah, D.N. Wade and K.M. Williams, Clin. Exp. Pharmacol. Physiol., 8, 653, 1981.

(2) A study of the distribution and elimination of high doses of phenytoin in rabbits. P.M. Chinwah, D.N. Wade and K.M. Williams. Clin. Exp. Pharmacol. Physiol., 8, 459-60, 1982.

(3) Macrodosage of phenytoin. G.G. Meredith, M. Kennedy, P.M. Chinwah and D.N.Wade. Med. J. Aust., 2, 584, 1979.

( 4) Multiple drug interactions with phenytoin. D. J. Birkett, G.G. Graham, P.M. Chinwah, D.N. Wade and J.B. Hickie, Med. J. Aust., 2, 467, 1977. (vi)

Other Publications

(5) Monitoring plasma concentrations of drugs. G.G. Graham, P.M. Chinwah, M. Kennedy and D.N. Wade. Med. J. Aust., 2, 124, 1980.

(6) Enhanced metabolism of mexiletine after phenytoin adminstration. E.J. Begg, P.M. Chinwah, C. Webb, R.O. Day and D.N. Wade. Br. J. Clin. Pharmacol., 14, 219-223, 1982.

(7) A pharmacological method of measuring mouth-caecal transit time in man. M. Kennedy, P.M. Chinwah and D.N. Wade, Br. J. Clin. Pharmacol., 8, 372-373, 1979.

(8) The measurement of mouth-caecal transit time using salicylazosulphapyridine. E. J. Begg, M. Kennedy, P.M. Chinwah and D.N. Wade. Clin. Exp. Pharmacol. Physiol., 7, 639-691, 1980.

(9) Comparison of enzyme immunoassay for gentamicin compared with other methods. P.M. Chinwah and K.M. Williams, J. Antimicrob. Chemotherap., 6, 561-562, 1980.

(10) Chlorbutol toxicity and dependence. T. Borody, P.M. Chinwah, G.G. Graham, D.N. Wade and K.M. Williams, Med. J. Aust., 1, 288, 1979.

(11) Crystalluria during flucytosine therapy. K.M. Williams, P.M. Chinwah and R. Cobcroft. Med. J. Aust., 2, 617, 1979. (vii)

ABBREVIATIONS

AUC area under plasma concentration versus time curve

BP blood pressure

CL clearance

EMIT enzyme immunoassay technique

HPPH p-hydroxyphenyl phenylhydantoin

ICG indocyanine green

GLC gas liquid chromatography

t 1 / 2 half-life of elimination

volume of distribution TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS (i)

ABSTRACT (ii)

PREFACE (Publications supporting this Thesis) (v)

ABBREVIATIONS (vii)

CHAPTER 1 INTRODUCTION

1 .1 General Introduction - Michaelis-Menten Kinetics 1.

1.2 The Absorption of Phenytoin 5.

1.3 The Disposition of Phenytoin 6.

1.3.1 Binding and Distribution 6.

1.3.2 Metabolic Clearance 9.

1.4 The Disposition of Phenytoin Following an 12. Overdose

1.4.1 General 12.

1.4.2 Absorption 12. 1.4.3 Binding and Distribution 13.

1.4.4 Metabolic Clearance 14.

1.5 Some Specific Factors Affecting the Metabolism 15. of Phenytoin

1.5.1 General 15.

1.5.2 Drug Interactions {Enzyme Induction) 18.

1.5.3 Effects of Alcohol on the Metabolism of 19. Phenytoin

1.5.4 Genetic Effects on Phenytoin Metabolism 20.

1.5.5 Effects of Liver Disease on Metabolism 20.

1.6 Aims of Present Study 21.

CHAPTER 2 MATERIALS AND METHODS

2.1 Materials 23.

2.1.1 Chemicals 23.

2.1.2 Instruments and Hardware 23. 2.1.3 Rabbits 25.

2.2 Sampling schedules 25.

2.2.1 Overdose Patients 25.

2.2.2 Rapid Metabolizers of Phenytoin 25.

2.2.3 Low Dose Pharmacokinetic Studies with 26. Rabbits

2.2.4 High Dose Pharmacokinetic Studies in 26. Rabbits

2.2.5 Blood Pressure Experiments in Rabbits 26.

2.2.6 Tissue Distribution Studies in Rabbits 26.

2.3 Analytical Methods and Procedures for Human 27. Studies

2.3.1 Enzyme Multiple Immunoassay Technique 27. (E.M.I.T.)

2.3.2 Overdose Patients 27.

2.3.3 Rapid Metabolizers of Phenytoin 28.

2.3.4 Alcohol Estimations 29. 2.3.5 Protein Binding Determinations 30.

2.4 Analytical Methods and Procedures for Animal 30. Studies

2.4.1 Low and High Dose Pharmacokinetics 30.

2.4.2 Phenytoin Tissue Distribution Studies 31.

2.4.3 The Effect of High Doses of Phenytoin on 32. Blood Pressure in Rabbits

2.4.4 Hepatic Blood Flow in Rabbits 33.

2.4.5 Estimation of Phenytoin Concentrations 34. Bile

2.4.6 Estimation of Phenytoin Concentrations 34. in Fat

2.4.7 GLC Assay for HPPH 35.

2.5 Pharmacokinetic Analysis of Data 35.

CHAPTER 3 RESULTS

3.1 Overdosed Patients 38.

3.2 The Pharmacokinetics of Phenytoin Elimination 48. in Rabbits 3.3 Distribution of Phenytoin into Tissues 56.

3.4 The Protein Binding of Phenytoin in Man and 69. Rabbits

3.5 Effects of Phenytoin on Blood Pressure in Rabbits 75.

3.6 Effects of Phenytoin on Liver Blood Flow in the 81. Rabbit

3.7 Rapid Metabolisers of Phenytoin 86.

3.8 Reliability of Phenytoin Estimations by E.M.I.T. 94.

CHAPTER 4 DISCUSSION

4.1 Phenytoin Overdosed Patients 99.

4.1.1 Apparent First Order Elimination Kinetics 99.

4.1.2 Apparent Saturable Kinetics 103.

4.1.3 Plateau Phase Elimination Kinetics 105.

4.2 Protein Binding 108.

4.3 The Effect of Phenytoin on Blood Pressure and 113. Hepatic Blood Flow

4.4 Rapid Metabolizers of Phenytoin 116. 4.5 Reliability of Phenytoin Estimations by E.M.I.T. 120.

CHAPTER 5 CONCLUSIONS 122.

6. REFERENCES 124.

7. methodo\09j addendu.-n 136 1.

1. INTRODUCTION

1 .1 General Introduction - Michaelis-Mente □ Kinetics

The pharmacokinetics of drug elimination are generally studied following single doses or less commonly during multiple dosage or infusion regimens. While there may be considerable variability of plasma elimination profiles observed between patients, the elimination of most drugs are usually modelled satisfactorily by first order kinetics. Accordingly, the rate of elimination (dAe/dt) is direc~ly proportional to the plasma concentration (C), the proportionality constant being the whole body clearance (CL). Thus,

dA0 /dt = CL . C ...... ( 1 )

However, the elimination of many drugs may involve enzymatic and active transport processes. Consequently, it may be anticipated that the rate of elimination drugs may be more realistically described by Michaelis Menten kinetics.

dAJdt = Vm 1 C/(Km 1 + C) + Vm2 C/(Km2 + C) + ...... Vmn. . + C/(Vmn + C) ...... (2).

where Vm 1 , Vm 2 ... Vmn are the maximal rate of elimination and Km 1,

Km 2 ... Km 2 are the apparent Michaelis constants of the various pathways. The apparent Michaelis constants are the plasma 2 .

concentrations at which pathways 1, 2, ..... n are functioning at 50% of their maximal capacities.

At low plasma concentrations, when C<

dA0 /dt = Vm1 C/Km1 + Vm2 C/Km2 + ...... + Vm 0 C/Km 0 •.• (3)

= CL.C ...... (5)

Thus, first order elimination is produced at low plasma concentrations whereas Michaelis-Menten kinetics may be seen at higher plasma concentrations.

Increasing doses and thereby increasing plasma concentrations of drug, will eventually lead to concentration independent elimination i.e. when C>>Km, equation (2) becomes

dA8 /dt = Vm 1 + Vm 2 + ...... + Vm 0 ...... (6)

In this situation the elimination occurs at a constant rate, independent of drug concentration. However, plasma concentrations must be greatly in excess of Km values for zero order kinetics to be attained. For example, for a drug with only one pathway of elimination, C must be 9 times Km to achieve 90% saturation of metabolic 3.

TABLE 1.

Km Values for Phenytoin

Km Reference

=_l_6.0 Grasela et al. (1983) 7.5* Blain et al. (1981) 9.4 Blain et al. (1981) 14.5 Atkinson and Shaw (1973)

* Data for children. All other data for adults. V max values are found in Table 4.1 .1 4.

elimination. At intermediate ranges of concentration, elimination follows mixed order kinetics - neither saturated nor first order.

First order kinetics are usually seen within therapeutic ranges of plasma concentrations, but Michaelis-Menten kinetics are seen with 3 major drugs even within plasma concentrations produced by moderate doses. These drugs are phenytoin (Arnold & Gerber, 1970), salicylate (Levy, 1973) and ethanol (Rang no et al, 1981 ).

The elimination of phenytoin and ethanol may be described by the simplified Michaelis-Menten equation (7)

dA8 /dt = Vm C/(Km + C) (7)

This equation is valid if there is one pathway of metabolism, although it has been suggested that other pathways may contribute to the clearance of both drugs. While it is now well known that phenytoin follows Michaelis-Menten kinetics, its elimination has frequently been described in terms of its half-life since over small ranges of concentration the kinetics may appear to be first order.

Work presented in this thesis is concerned with the elimination of large doses of phenytoin. Following a phenytoin overdose it is not uncommon to observe plasma concentrations of drug in the range 40 to 80 µg/ml. This is of the order of up to 15 times published values for Km (Table 1.). Consequently, the elimination of 5.

phenytoin following overdose is expected to observe Michaelis-Menten kinetics and indeed may even closely approach zero order kinetics.

1.2 The Absorption of Phenytoin

The absorption of phenytoin, as is the case with other drugs, is dependent upon the following factors: pKa and lipid solubility of the drug, pH of the media in which the drug is dissolving, its solubility in the medium and the amount of drug present and the rate of gastric emptying. These factors may be altered by the presence of certain foods or drugs in the intestinal tract or by the formulations employed.

Reported pKa values of phenytoin are 8.3 to 9.2 (Dill et al; 1956). Consequently, phenytoin is predominantly in the non-ionised form in the stomach and should be absorbed by passive diffusion. However, it is very insoluble (Woodbury and Swinyard, 1972) in gastric juice and this low solubility contributes to the slow absorption of phenytoin from the stomach.

On passage into the duodenum where the pH is approximately 7 to 7.5, more of the drug exists in the ionized form and hence phenytoin is more soluble in the intestinal fluid, although the solubility is still only 14 µg/ml at pH 7 (Woodbury and Swinyard, 1972). Increased solubility together with the large surface area and higher blood flow to the small intestine, allows absorption to take place more rapidly than in the stomach. It is at this site that the maximum absorption of 6.

phenytoin has been shown to occur in rats (Woodbury and Swinyard, 1972; Meinardi et al., 1975; Noach et al., 1958).

As a result of its poor solubility, the absorption of phenytoin following oral administration is slow compared with many other drugs. The half-life of absorption has been reported to range from 1.0 to 2.8 hours and there is marked intersubject variability with peak plasma concentrations generally occurring between 2.9 and 8.9 hours (Gugler, 1975). The rate of absorption may depend upon dosage. Large doses are more slowly absorbed than smaller doses, because of the longer time taken to dissolve the mass of drug in small intestine (Jung et al, 1970).

1 .3 The Disposition of Phenytoin

1 .3.1 Binding and Distribution

Phenytoin is rapidly and widely distributed throughout the body, with a distribution phase lasting less than 2 hours (Gugler et al., 1976; Glazko et al., 1969). Phenytoin concentrations which exceeded that of plasma have been found in liver, kidney, brain, skeletal. muscle and body fat of rats within 2 hours of intravenous admirwtration of the drug (Noach et al., 1958; Dill et al., 1956).

If phenytoin was distributed throughout tissues at a concentration equal to the free concentration in plasma, then the volume of distribution predicted by the model of Oie and Tozer (1979) is 1o 1/70 kg(0.14 I/kg) (Lunde et al., 1980). This value is considerably 7 .

lower than the actual volume of distribution and indicates that there is considerable binding of phenytoin to tissues.

The plasma binding of phenytoin is principally to albumin (Porter and Layer, 1975; Odar-Cedarlof and Borg a, 1966), although secondary low affinity sites on other proteins bind the drug when it is displaced from albumin (Monks et al., 1978).

Phenytoin is displaced from albumin by some acidic drugs such as salicylic acid, phenylbutazone (Lunde et al., 1970), halofenate (Karch et al., 1977) and valproic acid (Monks et al., 1978). The binding of phenytoin also is decreased in renal (Blum et al., 1972) and hepatic disease (Hooper et al., 1974).

Protein binding displacement should lead to an increase in the Vd of phenytoin such as has been demonstrated when phenytoin is

displaced by salicylate (Paxton, 1980; Fraser et al., 1980). The half-life of elimination of phenytoin was unchanged by salicylate since the protein binding displacement caused a proportional increase in clearance (Paxton, 1980). In contrast, the increased free fraction was associated with an increased volume of distribution and a decreased half-life in uraemic patients. This observation was attributed to enzyme induction by the uraemic state (Odar-Cedarlof and Borga, 1974). 8.

FIGURE 1,3.1

,.... GW ws PHe Pfto RB 31,'i 150 -E :J ...... -O"I 0 :$. t '-' C: C' .2 ,Q ~ ' ~ ~ II ~ g 100 25 ... 0 C u QI C: \I ·5 > C _gC 8 a. C e:, ~ ] Cl) 50 ::,. 11.5 C -a.Ill E ~ QI en- 100 -la) JOit 400 500 600 Dose of phen~oin (mg/day)

Relationship between serum phenytoin concentrations and daily dosage of the drug in 5 patients (Richens and Dunlop, 1975). The data illustrates a large variation in Vmax from approximately 150 mg/day up to greater than 600 mg/day. 9.

1.3.2 Metabolic Clearance

Phenytoin is cleared almost entirely by hepatic metabolism and the elimination rate follows Michaelis-Menten kinetics (Section 1.1 ). This capacity limited metabolism occurs within or below the '4o- lOOfA_mol/t.) therapeutic range (10-20 µg/ml) Asince Km values are quoted as 6.0- 14.5 µg/ml (Table 1). There is large intersubject variability in both Km and Vmax· The effects of saturable metabolism and the large variation in Vmax are illustrated in Figure 1.3.1 where the steady-state serum phenytoin concentrations are plotted against the dose of phenytoin for 5 epileptic patients (Richens, 1979).

lnterindividual variability in metabolic rates is also indicated by the wide range of reported half-lives. Half-life was found to range from 7-48 hours (Hooper et al., 1976) when the elimination of phenytoin was analysed assuming first order kinetics.

.. L L ,J)-5·r"e~lk~clAl\toil'\. 5· ( r- h.!J~o,9pnen..,. The major urinary metabolite,"(HPPH) is excreted as its glucuronide conjugate and accounts for 75% of the dose of phenytoin. It has been recently reported that the proportion of a single phenytoin dose excreted in urine as HPPH or its conjugate increased from the

first dose (74.9 ± 4.6%) to the second dose given 2 weeks later (79.3 ± 4.6%). This finding suggests that autoinduction of phenytoin metabolism may occur after relatively brief exposure to the drug. HPPH is also the only metabolite of phenytoin which has been found in plasma where generally it occurs at concentrations less than 1/10 of that of the parent (Woodbury and Swinyard, 1972). 10.

FIGURE 1,3,2

3

t10 OCH i 1 (Q_/~'-.. 0'1 1"0 osc__,__ttf 4 1) 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), the main metabolite in man (Butler, 1957).

2) A metabolite hydroxylated at the para position on both benzene rings, found in the rat (Woodbury, 1969).

3) A catechol metabolite, 5(3, 4-dihydroxyphenyl)-5-phenylhydantoin, found in the rat (Woodbury, 1969).

4) 5(4-hydroxy-3-methoxyphenyl)-5-phenylhydnatoin (Chang et al., 1972).

5&6) Diphenylhydantolc acid and - amino-diphenylacetic acid, possible metabolites described in early studies In man (Kozelka and Hine, 1943) but not confirmed since, though the former has been indentified in the rat (Chang, et al., 1970).

7) A dihydrodiol metabolite 5-(3, 4-dihydro>5y-1, 5-cyclo-hexadiene-1-yl)-5- phenylhydantoin, possibly produced in man (Atkinson, et al., 1970).

8) Meta isomer of HPPH, found In human urine in small amounts (Atkinson, et al., 1970). 11.

Small amounts of other metabolites have been identified in urine (Figure 1.3.2). These are 5-(m-hydroxyphenyl)-5- phenylhydantoin, diphenylhydantoic acid, 5-(3,4-dihydroxyphenyl)-5- phenylhydantoin, 5,5-di(p-hydroxyphenyl) hydantoin and catechol metabolites. Neither HPPH nor any of the minor metabolites have been shown to possess significant anticonvulsant activity. 12.

1.4 The Disposition of Phenytoin Following an Overdose

1.4.1 General

The pharmacokinetics of drug elimination following an overdose are often assumed to be similar to those observed after a therapeutic dose. The differences, however, may be considerable (Rosenberg et al., 1981) Massive amounts of ingested drug may saturate normal processes of distribution, tissue and protein binding and elimination.

Furthermore, data on the kinetics of drugs in patients following an overdose are often difficult to evaluate because of the unknown quantity of drug ingested, the uncertainty of the time of ingestion and because of intervening treatment such as gastric lavage and the administration of charcoal and emetics.

1.4.2 Absorption

As discussed previously the principal site of absorption of phenytoin is the small intestine and the amount of drug absorbed is largely dependent on drug solubility and its contact time with the small intestine. The solubility of phenytoin is such that not more that 100 mg can be expected to be in solution at any one time (Woodbury and Swinyard, 1972). Following an overdose, the problem is exacerbated, so much so that a slowly soluble mass of drug may form (bezoar formation). Schwartz (1976) has described a case of acute 13.

meprobamate poisoning which required a gastrotomy to remove a drug­ containing mass in order to prevent physical obstruction and further absorption. While bezoar formation has not been definitely demonstrated to occur with phenytoin, absorption might be expected to continue throughout the total gastrointestinal tract if an overdose is taken.

There are several reports describing prolonged periods following ingestion of a phenytoin overdose during which time plasma concentrations of phenytoin continued to rise or remained constant. In one case, a patient was admitted to hospital with a plasma phenytoin concentration of 56 µg/ml and was given a gastric lavage. Plasma concentrations continued to rise, however, to 97 µg/ml on the fourth day (Matzke et al., 1981 ). The authors attributed this phenomenon to formation of a large drug mass within the gastrointestinal tract, with consequent slow absorption. Similar observations attributed to delayed absorption have been reported by Gill et al. (1978) and Chaikin et al. (1979).

1.4.3 Binding and Distribution

Following a therapeutic dose of phenytoin, plasma protein and tissue binding sites are not saturated, unlike drugs such as disopyramide (Cunningham, 1977), salicylate, phenylbutazone (Aarbakke et al., 1977) and naproxen (Runkel et al., 1976) for which the proportional binding to plasma protein with increasing total concentration decreased within their therapeutic ranges. Ingestion of 14.

an overdose of phenytoin might be expected to lead to plasma drug (16 JA91rnl ) concentrations of 300 µmol/1 Awhich approach the concentration of albumin (600 µmol/1). The consequent increase in free drug concentration should result in relatively larger amounts of phenytoin being distributed to tissues and to the specific receptor sites. An increase in the volume of distribution might be expected, together with a disproportionate increase in drug action.

However, an increase in the free fraction of phenytoin apparently does not occur even at quite high total concentrations. Wilson et al. (1979) observed a case of prolonged toxicity lasting 13 days after an acute phenytoin overdose in a child. Plasma protein binding was determined on day 2 and day 8 when total phenytoin concentrations were 50 µg/ml (200 µmol/1) and 35 µg/ml respectively, and no change in the free fraction (8.5%) of phenytoin was observed over this range. Thus, the prolonged toxicity apparently resulted from slow elimination of DPH and was not a consequence of abnormal protein binding of the drug. While saturation of binding of phenytoin to circulating plasma proteins has not been found, it is still possible that saturation of binding to tissues cou!d still occur.

Cases of prolonged rises or plateau phases of drug plasma concentration-time profiles have been observed (section 1.4.2). Generally, these have been attributed to delayed absorption but redistribution from tissues to plasma has also been invoked as a mechanism of this phenomenom. Pruitt et al (1975) studied a patient whose plasma concentrations of phenytoin rose from 85 15.

µg/ml at 3 hours after cessation of phenytoin therapy to 108 µg/m I at 24 hours even though the patient was given a gastric lavage at 3 hours. In this case, the authors suggested that a possible explanation for the rise in phenytoin concentration was a redistribution of phenytoin back into the plasma compartment. However, since gastric lavage will remove the drug from the stomach only, continuing absorption from the small intestine might be expected. 1 6 .

1.4.4 Metabolic Clearance

As the dose of phenytoin is increased there is a change in the elimination kinetics from a first to a zero order process as has been discussed previously. Consequently, following an overdose of phenytoin, it is expected that elimination will closely approach zero order since plasma concentrations will be 30 µg/ml or more, considerably in excess of most reported Km values (Table 1). Zero order kinetics have been observed in a number of studies of phenytoin overdose (Atkinson and Shaw, 1973; Kutt et al., 1964; Garrettson and Jusko, 1974).

1.5 Some Specific Factors Affecting the Metabolism of Phenytoin

1.5.1 General

Drugs are primarily removed from the body by metabolism and renal excretion. The relative importance of each route depends on the drug and the capacity of the patient to eliminate the drug by that route. The major site of drug metabolism is the endoplasmic reticulum of hepatocytes, although it is becoming increasingly evident that other tissues such as white blood cells, placenta, skin, lung and especially the gut may play an important role in the metabolism of drugs. 1 7 .

TABLE 2

Drugs Which Increase the Elimination Rate of Phenytoin

Drug Reference

Carbamazepine Hansen et al, (1971) Christiansen and Dam (1973)

Ethanol Kater et al., (1969) Birkett et al., (1979) Baylis et al., (1971) Mattson et al., (1973) Furlanut et al., (1978) Makki et al., (1980)

Clonazepam Vajda et al., (1971) Diazepam Richens and Houghton (1975) Dichloralphenazone Riddell et al., (1980) Pyridoxine Hansson and Sillanpaa (1976) Phenobarbitone Kutt (1972) 18.

There are many factors which contribute to the interindividual distribution of metabolic clearance rates observed for most drugs. These include age, disease state, sex, renal, hepatic and cardiac functions. Of particular interest to the present study are those factors which lead to elimination profiles outside this normal distribution. These factors are drug interactions (enzyme induction) including the effect of alcohol on drug metabolism and inherited metabolic "defects" in drug biotransformation.

1.5.2 Drug Interactions (Enzyme Induction)

A number of drugs have been shown to enhance the metabolic elimination of phenytoin. (Table 2). Most of these changes are probably due to an increased metabolic clearance of phenytoin.

Although there is marked interindividual variation in the plasma half-life of phenytoin in man (Arnold and Gerber, 1970), blood concentrations remain stable for relatively long periods in patients (Kutt and Fouts, 1971 ). This indicates that despite the observation that phenytoin is a potent inducing agent, it apparently does not induce its own metabolism, or that induction is complete soon after commencement of treatment and during the accumulation phase (Dickinson et al., 1985). 19.

1.5.3 Effects of Alcohol on the Metabolism of Phenytoin

Drug overdoses are often accompanied by alcohol ingestion and alcohol can alter both the pharmacodynamics and pharmacokinetics of other drugs. The pharmacokinetic effects of alcohol are complicated by the finding that while acute alcohol ingestion may inhibit the mixed function oxidase system (Rubin et al., 1970) chronic alcohol exposure induces the metabolism of some drugs (Misra et al., 1971 ), although the development of cirrhosis leads to impaired elimination of drugs (Kater et al., 1961 ).

There is considerable evidence that the chronic ingestion of alcohol induces the metabolism of phenytoin (Table 2) Kater et al. (1961) have reported that when alcoholic and non-alcoholic patients were given phenytoin, the half-life was shorter in the alcoholic subjects. Birkett et al. (1979) have studied a patient with suboptimal plasma concentrations of phenytoin despite a daily intake of 600 mg. The low concentration of phenytoin was attributed to induction of its metabolism by chronic alcohol intake.

There is evidence, however, that chronic ethanol may not induce but inhibit phenytoin metabolism. Sandor et al. (1980) reported a significant increase in phenytoin clearance in 11 chronic alcoholics 1 week after cessation of alcohol; the author concluded that increased clearance of total and free phenytoin is probably due to the unmasking of inhibition of phenytoin biotransformation and not enzyme induction or changes in phenytoin free fraction. 20.

1.5.4 Genetic Effects on Phenytoin Metabolism

In many instances inherited metabolic defects have been associated with decreased enzymatic activity or complete absence of activity for specific essential metabolic transformations.

An inherited enzymatic defect which is not apparent in normal circumstances may become life threatening when a patient is challenged with a drug requiring that specific pathway for its metabolic clearance. A well known example is the effect of pseudocholinesterase deficiency on the activity of succinylcholine.

Brennan et al. (1968) studied 11 slow inactivators of isoniazid, 5 of whom developed diphenylhydantoin intoxication. This study suggested that the clinical problem of diphenylhydantoin intoxication arising in relation to isoniazid therapy is restricted to patients exhibiting a genetically determined slow inactivation for isoniazid.

1.5.5 Effects of Liver Disease on Metabolism

Plasma concentrations of phenytoin have been reported to increase as liver impairment increased (Kutt et al., 1964). This result is consistent with the liver being the major site of metabolism of phenytoin. 21.

Kutt et al. (1964) studied patients in whom tolerance of diphenylhydantoin and/or phenobarbitone decreased as liver impairment increased. Unmetabolized drugs accumulated in the body and a decrease in the output of metabolites was observed. This took place when commonly used dosages were admi~tered, although small doses (4-5 mg/kg) were usually metabolised adequately. Thus the "standard" dose was an overdose for these individuals. The authors concluded that findings were similar to those observed in patients with a genetically determined defect in diphenylhydantoin metabolism. Three of the four patients studied with lowered ceiling, chronic liver disease which was clinically diagnosed as cirrhosis associated with alcohol intake.

1 .6 Aims of Present Study

The elimination kinetics for phenytoin have been established in patients following normal therapeutic doses of these drugs, however, elimination profiles of phenytoin have received little attention when large doses are given.

The aims of the present study were:

1. To study the elimination profiles of phenytoin in man following phenytoin overdose.

2. To reproduce the observed profiles in an experimental animal, the rabbit. 22.

3. To examine the pharmacokinetic parameters which determine these profiles and in particular to study the distribution of phenytoin in rabbits following an overdose.

4. To study the elimination profiles of phenytoin in those patients requiring large doses (>600 mg/day) to reach adequate steady state plasma concentrations and to examine some pharmacological parameters which may contribute to the rapid elimination observed.

The data obtained from these studies may contribute to better management for those patients requiring high maintenance doses of phenytoin for control of seizures. Furthermore, data obtained by monitoring patients following a phenytoin overdose may be used to predict the time course of coma or other pharmaocological or toxic effects. They may also facilitate decisions such as whether to try to accelerate phenytoin removal by diuresis, haemodialysis or haemoperfusion, or whether to allow the body to clear the drug of its own accord and provide supportive treatment only. In any event, an understanding of the expected time course of elimination may shorten the period of patient hospitalization. 23.

2. MATERIALS AND METHODS

2.1 Materials

2.1 .1 Chemicals

E.M.I.T. reagents for the enzyme immunoassay of phenytoin and carbamazepine were obtained from Syva Company (Palo Alto, California).

Pure sodium phenytoin was donated by Parke Davis, Sydney. 14C-phenytoin was purchased from Amersham, Buckinghamshire, England.

Tissues were solublized using Protosol (New England Nuclear, Boston, U.S.A.). Hydrogen peroxide (AnalaR, BDH Chemicals, Australia) was the decolorizing agent. P.C.S. (Amersham, U.S.A.) was the scintillant solution.

2.1.2. Instrument and Hardware

The E.M.I.T. reaction was monitored at 340 nm with a digital reading spectrophotometer (Gildford 300T). It was equipped with a thermally regulated flow-cell which maintained the temperature at 30 ± 1o C. The spectrophotometer data output was processed by a Syva computer printer 1000. 24.

The perpex dialysis cells for protein binding work were supplied by the Chemical Rubber Company.

Phenytoin solutions were administered intravenously to rabbits via a Harvard Multifit Syringe pump (model 975), and samples were collected via an intravenous teflon 22G Surflo Catheter (Terumo Corp., Japan).

Blood pressure was monitored using a Harvard pressure transducer/recorder (model 350), calibrated against a mercury manometer.

Counting of radioactivity in tissues was performed on a Packard Liquid Scintillation Spectrometer (model 2650).

Estimation of phenytoin in fat and alcohol in blood were performed on a Hewlett-Packard (model 571 0A) Gas Chromatograph, fitted with flame ionization detector.

Measurements of indocyanine green for the clearance study were performed on the Pye Unicam 5P8-100 spectrophotometer at a wavelength of 800 mu. 25.

2.1.3 Rabbits

All rabbits used in the experiments were male, New Zealand white rabbits. They were allowed free access to food and water until the time of the experiment.

2.2 Sampling Schedules

Serial blood samples were collected from patients and rabbits into EDTA tubes. The plasma obtained from these samples was stored at ooc until assayed.

2.2.1 Overdose Patients

Blood samples (5 ml) were collected twice daily when possible for the duration of the patients stay in hospital and the actual times of collection were recorded.

2.2.2 Rapid Metabolizers of Phenytoin

The protocol involved the collection of serial blood samples (5 ml) at approximately the following times, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24 and 36 hours. Actual times were recorded. Selected samples were assayed during the course of the trial and collection of samples was terminated if plasma phenytoin concentrations fell below the sensitivity limits of the assay. 26.

2.2.3 Low Dose Pharmacokinetic Studies in Rabbits

Blood samples (0.5-1 ml) were taken at approximately the following times, 0, 5, 10, 15, 20, 30, 40, 50, 60, 80, 120, 150, 180, 240, 300, and 330 minutes. Actual times were recorded.

2.2.4 High Dose Pharmacokinetic Studies in Rabbits

Blood samples (0.5-1 ml) were collected at approximately the following times 0, 0.8, 0, 17, 0.25, 0,33, 0.5, 0.6, 0,8, 1.0, 1.3, 1.6, 2.0, 2.3, 2.6, 3.0, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 22, 23, 24, 25, and 26 hours. Actual times were recorded.

2.2.5 Blood Pressure Experiments in Rabbits

Blood samples (0.5 ml) were collected from 3 rabbits at various time intervals up to 7 hours after the infusion of the high doses (74 mg/kg) of phenytoin. Actual times were recorded.

2.2.6 Tissue Distribution Studies in Rabbits

Blood samples (0.5 ml) taken approximately at the following times, 0, 5, 10, 15, 20, 30, 45, 60, 80, 100 and 120 minutes prior to the sacrifice of the rabbits for tissue distribution studies. Actual times were recorded. 27.

2.3 Analytical Methods and Procedures for Human Studies

2.3.1 Enzyme Multiple Immunoassay Technigue (E.M.I.T)

Analyses of plasma phenytoin were performed in duplicate by the Enzyme Immunoassay Technique (Bastiani .el...fil., 1973, Rubenstein .et..all, 1972) (Commercial Kit, E.M.I.T., Syva, Australia).

In the E.M.I.T. assay, plasma is mixed with a reagent (Reagent A), which contains antibodies to a particular drug together with the substrates for the enzyme glucose-6-phosphate dehydrogenase (G6DPH). Binding occurs to a drug in the serum of plasma that is recognized by the antibody. A drug labelled drug combines with any remaining unfilled antibody binding site and the enzyme activity is thereby proportionately reduced. The residual enzymatic activity is directly related to the concentration of the drug present in the serum of plasma. The active enzyme converts nicotinamide adenine dinucleotide (NAO) to NADH, resulting in an absorbance change that is measured spectrophotometrically.

2.3.2 Overdose Patients

The 13 subjects in this study were selected from patients who were found to have highly elevated plasma concentrations of phenytoin on admission to hospital or during routine drug monitoring. In each case, patients were suspended from their drug therapy and blood specimens were collected in EDTA tubes at various intervals 28.

to establish an elimination profile. Phenytoin analyses in plasma were performed in duplicate by the E.M.I.T. technique (Syva).

Following a re-examination of the history of one patient (P.M.) it was decided that the original diagnosis of epilepsy was incorrect and phenytoin therapy was discontinued. A single dose (400 mg) of phenytoin was administered intravenously 3 months after cessation of the chronic phenytoin therapy. Blood samples were collected for analysis.

Half-lives were calculated by linear regression of the points on the drug plasma concentration time profile for those cases where elimination was apparently first order.

2.3.3. Rapid Metabolizers of Phenytoin

The subjects in this study were selected from patients found to have subtherapeutic plasma concentrations of phenytoin during routine monitoring and who remained subtherapeutic despite several increments in daily phenytoin dose above the normal 300 mg.

In each case, the patient was given a single 400 mg dose of phenytoin intravenously over 1 hour. Blood samples (5 ml) were collected into EDTA tubes at various intervals as described previously. 29.

Four of the 6 patients were classified as alcoholic on the basis of their medical histories. Blood levels of alcohol were determined on the initial blood sample from all subjects.

2.3.4 Alcohol Estimations

Alcohol estimations were performed on the first blood sample collected from all patients at the beginning of each study.

The technique involves a modification of the method by Hooper (1961 ). To the blood samples (1 ml) was added internal standard propan-2-o1 (1 ml, 30 mg/100 ml), sodium tungstate (1 ml, 10 g/100 ml) and sulphuric acid solution (1 ml, 0.6 N). The samples were mixed, allowed to stand for 5 minutes and centrifuged. An aliquot of the supernatant (5 ul) was injected into the gas-liquid chromatograph.

A standard curve was prepared by spiking sample of plasma with ethanol to give concentration ranging from 0.01 - 0.2 g/100ml. 30.

2.3.5 Protein Binding Determinations

Plasma samples containing phenytoin were spiked with 14C­ phenytoin (80 ul, 10 ug/ml) and dialyzed overnight against phosphate buffer (0.1 mol/I, pH 7.4) in 1 ml cells (Chemical Rubber Company). Washed celophane (Visking) was used as the dialysis membrane. Samples of dialysed plasma (0.5 ml) and dialysate (0.5 ml) were assayed by scintillation counting or by the gas chromatographic method described above.

The fraction of phenytoin which was bound (Fs) was calculated from

where Cct = number of counts in dialysate Cp = number of counts in plasma

2.4 Analytical Methods and Procedures for Animal Studies

2.4.1 Low and High Dose Pharmacokinetics

There were 3 rabbits in each group and their weights ranged from 2.25 kg to 3.35 kg. For the low dose pharmacokinetic studies, a single dose of 20 mg of sodium (equivalent to 18.4 mg of phenytoin) was administered irrespective of the rabbit's weight. For the high 31.

dose pharmacokinetics studies a single dose of 74 mg/kg of sodium phenytoin was given.

Sodium phenytoin was dissolved in distilled water (2 ml) by the addition of 0.1 ml of 2.5N NaOH and then the solution was adjusted to pH 10 by the addition of 3 ml of phosphate .buffer (0.1 M, pH 7.4). The solutions of sodium phenytoin were admin~tered through a teflon cannula into the marginal ear vein of the rabbit over 10 mins using a Harvard infusion pump. The blood samples were collected from the cannulated artery of the other ear. The arterial line was flushed with 0.5 ml of heparinized saline (1 O units/ml of sodium heparin in saline) after the collection of each specimen.

2.4.2 Phenytoin Tissue Distribution Studies

Sodium phenytoin was administered to 2 groups of 3 rabbits at a dose of 20 mg to the first group and 74 mg/kg to the second. Phenytoin infusion solutions were prepared as described previously but with the addition of 14C-phenytoin (0.268 mg).

The intravenous infusion of phenytoin was adminstered over 11 mins. The rabbits were sacrificed 2 hr after the end of the infusion and serial blood samples were collected from the cannulated ear artery until this time. The major organs, together with samples of fat from the fat pads and muscle from the thigh, were removed, rinsed with saline, blotted dry and weighed. All 32.

samples were homogenised using a Tyristor Regler homogeniser and were then stored at -2ooc until analysed.

Duplicate samples of the homogenates (approx. 20 mg) from each organ were weighed into glass vials and tissue solubilizer added (Protosol, 1 ml). The vials were heated (550C) overnight at which time all the tissue had dissolved. Hydrogen peroxide solution (0.5 ml, 30% v/v) was added to decolourise the samples and this was followed by addition of scintillant solution (PCS, 1O ml). All samples were counted for 10 mins in the scintillation counter.

2.4.3 The Effect of High Doses of Phenytoin on Blood Pressure in Rabbits

Phenytoin (74 mg/kg) was administered intravenously through a constant infusion pump over 10 minutes or 30 minutes to three rabbits on five occasions. The solution was prepared as previously described.

Blood pressure was monitored continuously for 12 hours directly from the cannulated artery using a pressure transducer.

Blood samples (0.5 ml) were collected at various time intervals over the course of the experiment. 33.

2.4.4 Hepatic Blood Flow in Rabbits

Hepatic blood flow was calculated by measuring the disappearance of indocyanine green from plasma according to the method of Caesar fil._gj_., (1961 ).

A single intravenous bolus injection of I.C.G. (0.5 mg/kg) was administered, and serial blood samples (2 ml) were collected at approximately the following times, 0, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, and 20 minutes. The actual times were recorded .

. The I.C.G. was admin'stered,. alone and then 4 hours later following a 10 min infusion of a low dose (18.4 mg) of phenytoin. The sample collection was as described previously. The rabbits were allowed to recover for one day before the infusion of a high dose (74 mg/kg) of phenytoin, again followed 4 hours later by a bolus of I.C.G.

The samples were assayed spectrophotometerically at 800 mu and the clearances were determined from the plasma concentration time curve. A standard curve was prepared from blank rabbit plasma spiked with I.C.G. to give concentrations ranging from 0.5 ug/ml to 15 ug/ml. 34.

2.4.5 Estimation of Phenytoin Concentrations in Bile

Samples of rabbit bile were analyzed directly using the E.M.I.T. technique in conjunction with an ABA 100 bichromatic analyzer (ABBOT).

A standard curve was prepared by spiking samples of blank rabbit bile with phenytoin to give concentrations from 0.5-30 ug/ml.

2.4.6 Estimation of Phenytoin Concentrations in Fat

Duplicate samples of fat (0.25 g) were dissolved in n­ hexane (10 ml), extracted with sodium hydroxide solution (2 ml, 1 N) and the organic phase discarded. The aqueous phase was then acidified with HCI solution (3 ml, 1 N) and extracted with ether (7 ml).

The ether was transferred to a conical tube and evaporated to dryness. The samples were resuspended into 1 ml of E.M.I.T. tris­ buffer (pH 7.9), and assayed as described for plasma phenytoin.

A standard curve was prepared from samples of fat spiked with phenytoin to give concentrations between 0.5 and 30 ug/ml. 35.

2.4.7 GLC Assay for HPPH

Plasma samples (1 ml) and standards in blank rabbit plasma (0, 10 and 20µg/ml) were spiked with internal standard (p­ toluylphenytoin, 20 µg) and hydrolysed with HCI solution (1 ml, 1ON). Samples were then neutralised with NaOH solution(1 ml, 1ON) and acidified with HCI solution (0.5ml, 1.0N). The HPPH was extracted with 10ml of ether. The organic phase was se'parated, evaporated under air and the residue taken up in NaOH solution (2.0ml, 0.1 N). This solution was washed with ether (1.0ml) and the ether discarded. The aqueous phase was acidified with HCI (1.0ml, 0.2N) and extracted with ether (1 0ml). The ether was then separated and evaporated to dryness. The residue was taken up in methylating agent (trimethylanilinium hydroxide, 0.1 N in methanol, 50µ1) and an aliquot was taken for analysis by GLC. Chromatographic conditions were: 3% OV-17 column, oven temperature 280°C, flow rate of the nitrogen carrier gas 60ml/min.

2.5 Pharmacokinetic Analysis of Data

The clearance (CL), volume of distribution (Vd) and half-life during the log-linear phase of the time course of plasma concentrations were calculated using the following relationships:

t112 = 0.693/B

CL = Dose/ AUC 36.

Vd = CUB

Where 13 is the slope of the terminal phase, and AUC is the area under the total time/plasma concentration curve. The AUC up to the last data point was calculated by trapezoidal rule and then extrapolated to infinite time.

The clearance and volumes of distribution were calculated assuming 100% availability since drug was adminstered intravenously.

.....i .....i

w w

75.2 75.2

59.2 59.2

58.0 58.0

56.5 56.5

55.2 55.2 71.5 71.5

S4.9 S4.9

53.5 53.5

66.5 66.5

43.6 43.6

72.0 72.0

!kg} !kg}

&S.0 &S.0

68.6 68.6

~ ~

Dose Dose

~00 ~00

300 300 300 300

3QQ 3QQ

300 300

300 300

300 300

300 300

300 300

300 300

400 400

400 400

400 400

{mg/day) {mg/day)

Phenytoin Phenytoin

Medications Medications

3,1,1 3,1,1

5-Fluro-

~o\e. ~o\e.

Thera12y Thera12y

diazepam, diazepam,

gentamicin gentamicin

and and

diazepam, diazepam,

B, B,

Concurrent Concurrent

Drug Drug

thioridazine thioridazine

and and

rne:th•o rne:th•o

TABLE TABLE

O"f O"f

carbamazepine carbamazepine

I I

Data Data

h h

nil nil

prednisone prednisone

phenobarbitone phenobarbitone metromidazole, metromidazole,

and and

phenobarbitone, phenobarbitone,

prednisolone, prednisolone,

carbamazepine carbamazepine

nil nil

nil nil

ampicillin ampicillin

nil nil

nil nil nil nil

chlonnethiazole, chlonnethiazole,

cystosine cystosine

amphotericin amphotericin

oxazepam, oxazepam,

Concurrent Concurrent

c c

Patient Patient

32 32

28 28

29 29

35 35

3Q 3Q

54 54

25 25

65 65

50 50

35 35

Age Age

20 20

33 33

G~ G~

M M

M M

F F

F F

M M

M M

f f

F F

F F

M M

M M

F F

M M

lli lli

w.c. w.c.

W.M. W.M.

G.A. G.A.

N.R. N.R.

R.B. R.B.

A.B. A.B.

J.P. J.P.

M.P. M.P.

J.C. J.C.

V.E. V.E.

T.L. T.L.

N.C. N.C.

L.J. L.J. ~ ~ 38.

3. RESULTS

3.1 Overdosed Patients

Twelve patients were included in this study and their daily maintenance doses of phenytoin were either 300 or 400mg. There were 6 males and 6 females. Their ages ranged from 20 to 65 years and 6 patients were known to be taking concurrent medications (Table 3.1.1 ). . Hospital records indicated that of the twelve patients, five had overdosed unintentionally as the result of either chronic therapy with an inappropriate dosage regimen or the patient had not followed the recommended dosage schedule. The remaining patients were epileptics who had ingested a single intentional overdose. No patients had a history of abuse of other drugs apart from alcohol .

There were four subjects (W.C., R.B., N.R. and N.C.) who were known to be alcoholics, again on the basis of information stated in the hospital records. Plasma ethanol concentrations were measured and they were found to be 0.02, 0.02, 0.11 and 0.05 g/100ml, respectively, at the time when the first blood sample was taken. The time of last ingestion of alcohol could not be determined. There was only one other patient (A.B.) who had detectable plasma ethanol concentrations. This patient was said not to be an alcoholic.

The overdosed patients could be divided into three groups according to the type of plasma concentration-time profile which was observed. Group 1 (Fig. 3.1.1) consisted of three patients in 39.

-100 1- ·,.. ' 0"- -~ 0 ---~ . z 0 10 t- N.c•-...... -v.E z> w i c( :E Cl) c( ..J Q. 1 50 100 150 200 250 300

TIME AFTER ADMISSION (hr)

Figure 3.1.1. Time courses of plasma concentrations of phenytoin in 3 patients demonstrating log-linear elimination of the drug. 40.

whom the decline in plasma concentrations was log-linear. The correlation coefficients for these log-linear plots were 0.971, 0.966 and 0.964.

Group 2 consisted of six patients in whom the elimination of phenytoin was not first order. Plots of the logarithm of plasma concentration versus time were clearly non-linear and showed marked downward curvature (Fig. 3.1.3). Plots of plasma concentrations versus time were approximately linear over the complete range of plasma concentrations found (Fig. 3.1.2). The initial plasma concentrations were 56.5 µg/ml (s.d.=13.8) falling to 6.8µg/ml (s.d.=5.8) at 5-10 days after commencement of the study. The slope of the linear plots of plasma concentration versus time allowed an estimation of the V max of the metabolism of phenytoin in these patients. The slopes ranged from 2.0-11.4 µg/ml/day.

Assuming a volume of distribution of 42 I, the V max values were calculated to range from 1.2 to 6.8 mg/kg/day. These values exceeded the daily dosage for 5 of the 6 subjects (Table 3.1.2).

In group 3 (Fig. 3.1.4) there were three patients who showed a plateau phase. These patients had initial phenytoin concentrations which remained at a "plateau" or continued to rise for some time after hospitalisation. For these subjects there was a period of 2 1/2 days (G.A.) and 7 days (N.R. and W.M.) during which time there was a plateau of phenytoin concentrations. In fact, for one patient (W.M.) the peak plasma concentration of phenytoin occurred 7 days 18 41.

_J ":c 78 C!I '::::, .,. V uz 0 68 u z H .... 0 I- >- z SIi l&I J: Q. 28 :c< Cl)

_J< II Q. a a Ill 128 161 TINE AFTER ADMISSION CHR> '

"_J lC -71 CD ':::, V u z 68- u0 g .. i SI f a

i II ~ • • ... tD 218 ZA 311 TIHE AnER ADHISSION CHR>

Figure 3.1.2 Time courses of plasma concentrations of phenytoin in 6 patients demonstrating ze·ro order elimination of the drug. Plots are approximately linear over the complete range of plasma concentrations. ,. 42.

.J" :E: a.itc~o C, ~c-..c ::,' V u li8 z 0 ■------■ "'"D u A ---~~ 0 ~z ~L.Z. la.I X A. < :E: •,1'.B. U) <_, :J:"P. A. 18 8 48 88 128 UII TINE AnER ADNISSION CHR>

188

I.I ___...... _ ___...__ __--'------'----=1----'----..J

8 68 188 l&8 211 258 TI~ AnER ADHISSICH CHIO

Figure 3.1.3 Semi-logarithmic plots of plasma concentrations of phenytoin in 6 patients demonstrating zero order elimination of the drug. Note the marked downward curvature in contrast to the linear plots in Figure 3.1.2. 43.

100

t 50 ~- ~ 8 z ez w \ :c A. 4( ~ !Q w.\ ..I A. 10 5 10 15 20 TIME AFTER ADMISSION (days)

_ 100 'i...... • --• 0z ~··-•··-· ...... 0 0 -z OI- 10 >z w a.:::z:: < :E en < ~ 1------·40 80 120 160 200 TIME AFTER ADMISSION (llr)

Figure 3.1.4 Time courses of plasma concentrations of phenytoin in 3 patients showing plateau phases. 44.

TABLE 3.1.2

Maximal Rate of Phenytoin Elimination from Subjects with Zero Order Elimination

Subject Max. Elimination Rate Phenytoin Dose r2 µg/ml/h µg/ml/d mg/kg/d mg/kg/d

M.P. 0.202 4.9 2.9 4.5 0.992 A.B. 0.085 2.0 1.2 6.9 0.931 R.B. 0.474 11.4 6.8 5.4 0.990 L.J. 0.359 8.6 5.2 7.1 0.998 J.T. 0.274 6.6 4.0 5.6 0.999 J.P. 0.215 5.2 2.6 5.2 0.997 45.

"' 10 -I ~ (!) :::>' V

8 s 18 IS 28 25 TIME CHRS)

Figure 3.1.5 Log plasma concentration-time profile of phenytoin in the patient G.A. following administration of a single 400mg dose of drug. 46.

after the overdose and did not fall beneath the initial plasma concentration for 14 days. Following the plateau there was a relatively precipitous drop in plasma concentrations, which were approximately log-linear with an estimated mean half-life of 22.4 (s.d.=3.9) hours.

The patient G.A. was rechallenged with a single dose of phenytoin on a later date. Figure 3.1.5 shows the patient's phenytoin elimination profile which had a half-life of 17 hours which is within the normal range. 47.

TABLE 3.2.1

Phenytoin Elimination Parameters in the Rabbit Low Dose (18.4 mg)

Rabbit .Ql AUC (ml/min) (ktQ, h ml-1)

1 21.7 3.67 1.95 14.7 2 10.2 2.35 2.67 31.4 3 11.6 3.22 3.21 27.5 5 23.8 2.66 1.29 12.9 6 16.3 2.01 1.42 18.9 7 26.0 2.29 1.02 11.8 Mean 18.3 2.70 1.93 19.5 so 6.6 0.63 0.86 8.2 s.e.m. 2.7 0.26 0.35 3.3

3 (27 .2 mg/kg) 13 .2 3.97 120.0 48.

3.2 The Pharmacokinetics of Phenytoin Elimination in Rabbits

A commercial solution of sodium phenytoin (Parke-Davis) was used in initial pharmacokinetic studies in rabbits. However, although this proved satisfactory for low dose pharmacokinetic investigations, 4 rabbits died within 5 hours following administration of 74 mg/kg of sodium phenytoin using the Parke Davis formulation. It was noted that blood collected from these rabbits was grossly haemolyzed and it was thought that haemolysis may have been caused by the propylene glycol in the Parke-Davis formulation. Subsequently, phenytoin was administered as an aqueous solution buffered to pH 11.0. Blood samples collected from these rabbits were not haemolyzed and no rabbits died.

The plasma elimination profiles of phenytoin were studied in a total of 7 rabbits following intravenous administration of either 18.4 mg (total), 27.2 mg/kg or 74 mg/kg of phenytoin.

After admin~tration of 18.4 mg of phenytoin, there was a rapid distributional phase (less than 1 .0 hr) followed by a log-linear phase in which the half-life was 1.9 hr (s.e.m.=0.4; Table 3.2.1 ). Examples are shown in Figure 3.2.1. There was no evidence of saturation of metabolism under these conditions, since the elimination phase was log-linear. The peak plasma concentrations immediately following infusion were between 6 and 9 µg/ml and the concentrations were monitored down to approximately 2 µg/ml. 49. " 18 ..J ::c (!) ::>' V +++ Z 6 + + 0 H t­ < t-°'z l&J u z + 0u :c< Cl) < _,1,___ __. ____ .&.... __ __, a...J ,.a .______...... ___

e e.5 1 .5 2 2.5 3

" ta ..J 0 ::c 00 (!) ::,' 0 0 V 0 0 Z 6 0 H t- «< t-z l&J u z 0u ::c< Cl) < ___..,__ ___ ..,______..J- ___..J a....J 1.0 ,.____ .._

e 2 ◄ s e

" 18 ::c...J a'(!) •••• z 6 0 H t- «< t-z w u z 0u <::c Cl) < et , .e ~-"---"---""'--""'--..,__ _.__..&..._-'----'----'---'---" e e.5 1.5 2 2.S 3 3.S ◄ ◄ .5 6 6.S 8 TJ:NE CHRS) Figure 3.2.1 Log plasma concentration-time profiles of phenytoin concentrations following a bolus intravenous dose (18.4 mg) of drug to rabbits 1, 2 and 3, respectively. 50.

taaa -I "%: sea (!) :::,' V ~ ,ea t-t t- < 6a 0:: • zt- •••• l&Iu ••• z 18 • 0 u 6 <%: (I) < -I a. 1.8 a e a 12 16 IS 21 24 TINE CHRS:>

"tea -I %: (!) 68 CtJ C :::,' C □ V a z 0 t-t □ a t- a < 0:: 18 t-z L&J u z 6 0 u a < %: (I) < -I Q. 1.8 a a 12 IS IS 21 24 27 TINE CHRS:>

Figure 3.2.2 Log plasma concentration-time profiles of phenytoin in rabbits #1 and #2, respectively, following administration of a large single bolus intravenous dose (74 mg/kg) of drug. 51.

A single rabbit given a 27.2 mg/kg dose of phenytoin (Rabbit 3·5 3, Table 3.2.1) had a somewhat longer half-life of -9-:& hours but no there was no evidence of Michaelis-Menten kinetics from the time course of plasma concentrations.

As no saturation of metabolism was evident at these doses of 18.4 mg (Fig. 3.2.1) and 27.2 mg/kg , dose was increased further to 74 mg/kg (approximately 200 mg for a 3 kg rabbit). At this dose, plasma concentrations at first fell rapidly for a short period (15-30 mins) from a mean of 79 µg/ml (s.e.m. = 10) but then reached a plateau phase (mean 49 µg/ml s.e.m = 3) for up to 2.2 hours (mean =1.8, s.e.m. = 0.2). In some instances, phenytoin concentrations even increased (Figs. 3.2.2 - 3.2.3).

Following the plateau, the plasma concentrations declined rapidly with a mean terminal half-life of 3.6 hours (s.e.m.= 0.7) hours which slightly exceeded that observed following the low dose (18.4 mg/kg).

The plasma phenytoin elimination profile in one rabbit was determined by measuring both total radioactivity and phenytoin alone (Fig. 3.2.4). Plasma concentrations of HPPH were also measured directly by GLC. This data showed that the difference between the phenytoin concentration and the measured total radioactivity was entirely accounted for by the sum of HPPH and phenytoin. Consequently, HPPH is the only significant metabolite in plasma. There were significant fluctuations in the phenytoin 52.

z 0 H + eet­ + ~ 18 zt- + l,J ++ ~ 6 + 0u + < J:: (I) < a....J I.a ___.____. __ __., _ _,__.....__...... __ _._ _ _._ ___...__..,.._.___. __ __.

a 2 ◄ 6 e 7 8 8 18 11 12 13 I ◄ TIME CHRS)

" iaca ..J J:: (!) 68 :)' V 0 z 0 0 H 0 t- 0 < 0 ~ 18 t-z l,J u z 6 0 Q u0 < 0 J:: Cl) < ..J a.. t.a IGI II 12 a 2 :, ◄ 6 6 7 a TIME CHRS)

Figure 3.2.3 Log plasma concentration-time profiles of phenytoin in rabbit #4 following administration of a large single bolus intravenous dose (74 mg/kg) of drug on two separate occasions. 53.

}- 100 z 0

a:ti zt­ w ~ 0 (.) cc :E Cl)cc ..J Q.

1..__--.___.___ ...._~~---~~~~-~':":"'-:'":---:"! 2 4 6 8 10 12 14 16 18 20 22 24 26

TIME Or)

Figure 3.2.4. Log plasma concentration-time profile of phenytoin (•) and HPPH (*) and "phenytoin" (*) concentrations based on measurent of total radioactivity following administration of a bolus intravenous dose (74 mg/kg) of 14C-labelled phenytoin to a single rabbit. 54.

concentrations measuring total radioactivity. These erratic concentrations appear to be real as they were also observed for HPPH which was measured independently in plasma by G.L.C.

The highest concentration of HPPH (approximately 20 µg/ml) was observed in the first plasma sample collected i.e. immediately following the 10 minute infusion of the high phenytoin dose. This concentration was approximately 17% of the phenytoin concentration at that time. At 21 hours, the plasma HPPH concentration was 5 µg/ml at which point, HPPH was 80% of the phenytoin concentrations. 55.

0 a.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 2 2.2 TIME CHRS)

Figure 3.3.1. Typical log plasma concentration-time profile of phenytoin following administration of a single bolus intravenous dose (18.4 mg) of radiolabelled ·drug to a rabbit (#6) prior to sacrifice for the study of the distribution of phenytoin into tissues. 56.

3.3 Distribution of Phenytoin in Tissues

The distribution of phentyoin was examined in two groups of three rabbits. The groups received either 18.4mg or 74 µg/kg of 14 C­ labelled phenytoin and the animals were killed 2 hours after dosage. The mean phenytoin plasma concentration at sacrifice in the low dose study was 2.6 µg/ml (s.d.= 0.8) and in the high dose study it was 48.1 µg/ml (s.d.= 1.5). These profiles (Figs. 3.3.1 and 3.3.2) were very similar to those observed in the rabbits used in the pharmacokinetic studies.

There was extensive tissue distribution of the drug for both high and low doses as determined by scintillation counting of tissue homogenate (Table 3.3.1). The levels of phenytoin were higher than plasma in well perfused tissues (lung, liver, kidney), although myocardium had a distribution ratio of 0.9. There was a high proportion of radioactivity in urine (ratio 34.9), bile (13.1) and bowel contents (5.5). There was a slight preferential distribution into fat (1.2) and bowel wall (3.4).

As a percentage of the total counts phentyoin was distributed in decreasing order to blood > bowel contents > fat > muscle. Although the relative distribution to muscle is low (0.5) the total mass of muscle is large (300 g/3.5 kg rabbit) and consequently, muscle accounted for aproximately 5% of the dose. 57.

" tee .J::c (!) ::::>' • '-' ■ z 60 ■ ■ • • 0 ■ • H • t- • < t-°'z ~ uz 0 u < ::c U) < ...J a. ua e 0.2 -0. 4 0.6 0.8 1.2 1. 4 1.6 1.8 2 2.2 TIME CHRS)

Figure 3.3.2. Typical log plasma concentration-time profile of phenytoin following administration of a single bolus intravenous dose (74 mg/kg) of drug to a rabbit (#8) prior to sacrifice for the study of the distribution of phenytoin into tissues. 58.

There were 3 estimates used for calculating the data for muscle, plasma and blood. (Mituka and Raunsley, 1968)

1) 300 g muscle for a large rabbit (3.5 kg). 2) Blood content of 65 ml/kg and 3) Haematocrit of 42%.

The fat content was the weight of fat (109 g) disi~ted from a single 3.1 kg rabbit.

The total recovery of radioactivity varied between 48% and 88% (mean 69.8%). A comparison of recovery of phenytoin from fat, bile and gut contents was made using radioactivity and direct measurement and the data are shown in Table 3.3.5, 3.3.6 and 3.3.7, ph4-n._vtoin respectively. Fat, bile and gut contents were analyzed for phen.tyoir by the EMIT assay (Tables 3.3.2, 3.3.3, 3.3.4, respectively). These results show that the radioactivity in fat is almost all phenytoin whereas that in the gut contents and bile is primarily phenytoin metabolite. Similarly, the radioactivity in urine was shown to be primarily metabolites. The recovery of the total radioactivity in urine at 2 hours, was 42.8% for the low dose, as compared to only 5.9% following the high dose of phenytoin. 59.

TABLE 3.3.1

THE DISTRIBUTION OF PHENYTOIN TO TISSUES IN RABBITS {Mean and S.D.)

TISSUE LOWOOSE HIGH DOSE

TissueLPlasma 0/q TQtal TissueLPlasma 0/q TQtal RatiQ

Plasma 1.0 5.4 (0.5) 1.0 5.6 (3.1)

Blood 1.0 8.9 (1.1)

Liver 2.4 (0.3) 6.2 (1.0) 2.3 (0.2) 7.3 (2.4) Heart 0.9 (0.2) 0.3 (0.1) 1.5 (0.5) 0.5 (0.2) Lung 1.9 (1.1) 0.9 (0.6) 2.5 (1.5) 0.9 (0.4)

Kidney 2.6 (0.9) 1.6 (0.3) 2.8 (0.3) 2.0 (0.7) Fat 1.2 (0.2) 5.3 (0.7) 2.9 (0.6) 14.5 (5.0)

Muscle 0.5 (0.1) 4.9 (0.5) 0.6 (0.2) 6.8 (2.0)

Brain 0.8 (0.1) 0.3 (0.1) 1.1 (0) 0.5 (0.3)

Stomach 0.8 (0.1) 0.5 (0.1) 1.1 (0.1) 0.7 (0.3)

Stomach Contents 0.3 (0.2) 0.3 (0.3) 0.1 (0) 0.2 (0.1)

Small Intestine 1.6 (0.5) 2.5 (0.5) 1.2 (0.8) 2.5 (1. 7)

Caecum 0.5 (0.1) 0.8 (0.1) 0.7 (0.2) 1.0 (0.3)

Colon 0.7 (0.1) 0.4 (0.1) 0.9 (0.1) 0.6 (0.3)

Rectum 0.6 (0.3) 0.4 (0.1) 0.8 (0.2) 0.6 (0.1)

S. Intestine Contents 3.6 (1.4) 2.9 (2.3) 2.1 (1.6) 3.2 (1.5)

Caecum Contents 0.9 (0.3) 5.7 (2.5) 0.2 (0.1) 1.5 (0.7)

Colon Contents 0.5 (0.2) 0.3 (0.1) 0.4 (0) 0.2 (0.1)

Rectum Contents 0.5 (0.2) 0.2 (0.2) 0.4 (0.1) 0.2 (0.1) 60.

TABLE 3.3.1 (continued)

TISSUE LONDOSE HfGHDOSE Tissue/Plasma % Total Tissue/Plasma % Total Ratio (gig} Counts Ratio (gig} Counts

Bile 13.1 (5.2) 0.7 (0.6) 7.8 (2.5) 0.3 (0.1)

Spleen 0.8 (0.2) 0.1 (0.1) 1.6 (0.8) 0.03(.04)

Testes 0.8 (0.2) 0.2 (0.2) 1.1 (0.4) 0.5 (0.3)

Urine 34.9 (24.0) 31.9 (11) 7.3 (0.9) 13.2 (0.9) 61.

TABLE 3.3.2

Distribution of Phenytoin into Fat

Rabbit No. Phenytoin Cone. Total Phenytoin % Total (µg/g) (mg)* Dose*

Low Dose 5 8.9 1 .1 5.5 (18.4 mg) 6 13.1 1 .1 5.5 7 8.9 1 .1 5.5 mean 10.3 1 .1 5.5

High Dose 8 421.4 41.4 20.0

(74 mg/kg) 9 323.7 30.6 16.0 10 215.5 19.0 10.2 mean 320.2 30.3 15.4

* From measurements of radioactivity. 62.

TABLE 3.3.3

Distribution of Phenytoin into Bile

Rabbit No. Phenytoin Cone. Total Phenytoin % Total (µg/m I) (µg) Dose

Low Dose 5 7.2 22.3 0.12 (18.4 mg) 6 8.6 12.1 0.06 7 * * mean 7.9 17.1 0.09

High Dose 8 55.5 138.9 0.07 (74 mg/kg) 9 37.6 26.9 0.01 1 0 71 .8 86.2 0.05 mean 54.9 84.0 0.04

* No bile recovered. 63.

TABLE 3.3.4

The Distribution of Phenytoin into Gut Contents

Section of GI Total Phenytoin Distributed to the Gut Contents (µg) Low Dose High Dose (18.4 mg) (74 mg/kg)

Rabbit No. 9 10 11 12 13 14 Stomach * * 39 40 * 62.0 Small Intestine 16.2 48 83 894 1047 1011 Caecum 85.0 342 * 709 371 1238 Colon 17.7 * * 49 8 118 Rectum * * * 8 * *

Total 119 391 122 1700 1425 2429 % of Dose 0.06 2.0 0.6 0.8 0.7 1.3 64.

TABLE 3.3.5 Recovery of Phenytoin From Fat Comparison of Radioactivity and Direct Measurement Determination

Rabbit No. Phenytoin Recovery (%) Radioactivity Chemical Assay

Low Dose (18.4 mg) 5 5.6 5.5 6 5.9 5.5 7 4.5 5.5 mean (sem) 5.3 (0.4) 5.5 (0)

High Dose (74 mg/kg) 8 19.3 20.0 9 15.0 16.0 10 9.4 10.2 mean (sem) 14.6 (2.9) 15.4 (2.8) 65.

TABLE 3.3.6 Recovery of Phenytoin From Bile Comparison of Radioactivity and Direct Measurement Determinations

Rabbit No. Phenytoin Recovery (%) Radioactivity Chemical Assay

Low Dose (18.4 mg) 5 1.17 0.12 6 0.36 0.06 7 * *

High Dose (74 mg/kg) 8 * 0.07 9 0.16 0.01 10 0.41 0.05 66.

TABLE 3.3.7 Recovery of Phenytoin From Gut Contents Comparison of Radioactivity and Direct Measurement Determination

Rabbit No. Phenytoin Recovery (%) Radioactivity Chemical Assay

Low Dose (18.4 mg) 5 10.9 0.6 6 7.0 1.9 7 10.8 0.6 mean (sem) 9.6 (1 .3) 1 .0 (0.4)

High Dose (74 mg/kg) 8 8.1 0.8 9 4.1 0.7 10 3.9 1.3 mean (sem) 5.4 (1.4) 0.9 (0.2)

Ratio Phenytoin/HPPH Low Dose High Dose 0.11 0.17 67.

TABLE 3.3.8

Urinary Recovery of HPPH From o to 2 Hours After Phenytoin Administration

HPPH Qonc. Urine Volume Recovered 0/Q of Total (µg/ml) !.mll. .(mgl ~

~igh Dose Rabbit

8 520 22 11.4 5.5 9 188 54 10.2 5.2 10 281 47 13.1 7.1

Low Dose Rabbit

5 297 41 12.1 63.1 6 181 44 7.9 41.4 7 463 10 4.6 23.9 68.

Table 3.4.1.

Effect of Plasma Concentration on Binding of Phenytoin in Overdosed Patients.

Patient Toxic Plasma Cone, Free Phenytoin Therapeutic Plasma Free Phenytoin

(µg/ml) 00 Cone. (ugtml) !%.

N.C. 42.0 µg/ml 13.3 12.0 12.5 w.c. 40.0 µg/ml 16.3 13.0 15.8

V.E. 30.0 µg/ml 13.4 6.0 14.6

N.R. 51.0 µg/ml 13.3 6.3 13.9

W.M. 45.0 µg/ml 13.3 14.0 13.8

G.A. 50.4 µg/ml 13.9 13.4 13.6

A.B. 32.0 µg/ml 13.4 4.0 11.6

J.P. 65.0 µg/ml 15.2 10.0 13.6

L.J. 57.6 µg/ml 12.9 10.9 15.9

M.P. 57.8 µg/ml 13.6 12.0 13.3

J.T. 43.8 µg/ml 14.4 0.2 11.6 69.

3.4 The Protein Binding of Phenytoin in Man and Rabbits

Protein binding was examined in the 11 patients who had been overdosed with phenytoin. Free phenytoin fraction determined on specimens collected from the patients on admission were not significantly different from those observed when plasma concentration had returned to the therapeutic range (Table 3.4.1.). There was no significant difference between the plasma protein binding of phenytoin in alcoholic and non-alcoholic subjects, although there was a small increase in unbound phenytoin over the range of 10 to 100 µg/ml (Table 3.4.2).

It was found that HPPH at concentrations from 1 to 100 µg/ml did not displace phenytoin at 10 µg/ml (Table 3.4.3). The different proportions unbound in Tables 3.4.2 and 3.4.3 are the result of interpatient differences in the degree of protein binding.

There was no significant effect of heparin or EDTA on the plasma protein binding of phenytoin at total phenytoin concentrations of 10 µg/ml (Table 3.4.4). The effects of freezing and thawing on the plasma protein binding of phenytoin was also examined. Fresh plasma had a mean of 11.5% free phenytoin (total phenytoin concentration 10 µg/ml) while following freezing and rethawing there was 12.8% free phenytoin (Table 3.4.4).

There was a marked effect of increasing concentrations of phenytoin on protein binding in rabbit plasma (Table 3.4.5). The in 70.

vitro results for the binding of phenytoin in rabbit plasma were confirmed by the limited in vivo data. Thus, a single rabbit given a high dose of phenytoin had 8.8% free phenytoin 2 hours after the infusion, but 2 hours following a low dose infusion there was 7.0% free phenytoin. The total plasma concentrations in this rabbit at 2 hours were 49 and 5 µg/ml, respectively. This difference is in agreement with the in vitro data (Table 3.4.5). 71.

TABLE 3.4.2

The Effect of Concentration of Phenytoin on its Binding to Human Plasma Proteins in vitro

Phenytoin Concentration Free Phenytoin (µg/m I) 00

10 12.6 20 13.2 30 12.8 40 13.4 50 13.3 100 13.8 72.

TABLE 3.4.3

Effects of HPPH on the Binding of Phenytoin to Human Plasma Proteins in vitro

Cone. HPPH (µg/ml) % Phenytoin Free*

0 8.2 5 8.0 10 8.5 100 8.6

* determined at a total phenytoin concentration of 10 µg/ml 73.

TABLE 3.4.4

The Effect of Anticoagulants on the Binding of Phenytoin to Human Plasma Proteins

Anticoagulant+ Free Phenytoin (%)*

None (plain clotted tube) 12.8 Heparin 14.4 EDTA 13.3

+ as contained in Venoject tubes *determined at a total phenytoin concentration of 10 µg/m I mean of duplicate determinations 74.

TABLE 3.4.5

Effects of Increasing Concentrations of Phenytoin on Protein Binding in Rabbit Plasma

Phenytoin Concentrations Free Phenytoin (µg/ml) (%)

100 12.3 80 11.7 60 10.1 40 9.6 20 10.4 10 7.8 5 7.8 75.

3.5 The Effect of Phenytoin on Blood Pressure in Rabbits

The mean blood pressure-time profiles for three rabbits infused with the large dose of phenytoin (74 mg/kg) on 5 occasions are shown in Figures 3.5.1 to 3.5.3. On four occasions, phenytoin was administered over 10 minutes, but was infused over 30 minutes in one experiment. There was no clear difference between the blood pressure response observed after the 30 minute and the 10 minute infusion schedules. Consequently, data from the five experiments have been grouped to determine mean values. During the infusion, mean blood pressure fell from 85 mm Hg (s.e.m. = 9) to 64 mm Hg (s.e.m. = 6), then immediately following the infusion rose rapidly to a mean blood pressure of 106 mm Hg (s.e.m. = 9), at which time the rabbits had generalised seizures. The mean blood pressure then declined steadily to a mean plateau of 68 mm Hg (s.e.m. = 4) over a period of approximately 2 hours (Table 3.5.1). Changes were also seen in heart rate. The infusion of the high dose of phenytoin decreased the heart rate from 200/min to 165/min. The rate then returned to normal over a period of approximately 4 hours.

Infusion of a saline control (Figs. 3.5.2) or the low dose of phenytoin (18.4 mg) did not alter the blood pressure or heart rate. 76.

tee ,n..,._,$tAAT ae

"Q 8e ~ e e V . a. 79 en z ee ~ l:

60

0 20 <40 60 80 100 120 i-40 use 180 200 220 TIME CMINS)

1aa

Qlil

8ta :c"'Q) E 'E 70 V'

Q. m 60 z < l&J :I: 60

40

39 0 za 49 ea sa 1 rara 12a 1 40 , era usra 290 22111 24111 TIME CMINS)

Figure 3.5.1. The mean blood pressure-time profiles following infusion of a large dose (74 mg/kg) of phenytoin to the same rabbit (#12) on two occasions. 77.

12111

"'a, :I: 1111111 stta..t E 'E V

Q. en ea z w< t :!: E..d pi«"Y'h,i"' 6111

◄ Ill

111 2111 ◄ 111 era era 1111111 12111 1 ◄ 111 1e111 1s111 2111111 22111 2 ◄ 111 2e111 TIME CMINS)

1 ◄ 0

120

"Q ~ 100 e 'e V . 80 Q. Begi" i 541,,_e. End 0:l 1''-'fibi"' z 60 w< ::c

40

20 0 20 40 60 80 100 120 140 169 180 200 220 240 260 TIME CHINS)

Figure 3.5.2. The mean blood pressure-time profiles following infusion of a large dose (74 mg/kg) of phenytoin to the same rabbit {#13) on two occasions. Also illustrating the absence of effect of a saline infusion on mean blood pressure. 78.

1:,a

"Q) :I: 110 e 'e V

Q. 90 al z < I.LI:c 70

0 s0 1 00 150 200 250 :,00 :,50 -400 -450 500 550 TIME CMINS)

Figure 3.5.3. The mean blood pressure-time profile following infusion of a large dose (74 mg/kg) of phenytoin to a rabbit (#14). 79.

TABLE 3.5.1

Mean Data for the Effect of Phenytoin on Blood Pressure in the Rabbit

Blood Pressure (mm Hg)

High Dose* Low Dose# Saline# Before Infusion 85 (s.e.m. = 3) 88 94 End Infusion 64 (s.e.m. = 6) 84 94 Immediate Post Infusion 106 (s.e.m. = 9) 88 100 Plateau 68 (s.e.m. = 4) 88 95

# n = I 80.

TABLE 3.6.1

The Effects of Phenytoin on the Pharmacokinetic Parameters for I.C.G. in Rabbits (mean and s.e.m, n=4)

Parameters Untreated Phenytoin Phenytoin (18.4 mg) (74 mg/kg) Cl (ml/min) 16.2 (0.6) 22.2 (0.8) 14.11 (2.8) Vd (ml) 320 (20) 360 (30) 270 (60) t1,2 (min) 13.8 (1.1) 10.2 (0.5) 12.8 (0.4) 81.

3.6 Effects of Phenytoin on Liver Blood Flow in the Rabbit

The effect of low and high doses of phenytoin on I.C.G. clearance were studied in 3 rabbits (Figs. 3.6.1-3.6.3). An increase in the systemic clearance of I.C.G. was observed after administration of the low dose of phenytoin and a small decrease in systemic clearance after the high dose (Table 3.6.1 ).

The increase in plasma I.C.G. clearance led to a decrease in I.C.G. elimination half-life after 18.4 mg phenytoin. Following treatment with 74 mg/kg of phenytcin there was a decrease in the I.C.G. elimination half-life associated with the decrease in I.C.G. clearance (Table 3.6.1 ). 82.

I"\ 10 ..J ::c (!) :J' + V 0 □

0 0.1 0.2 0.3 0.4 e.s TIME CHRS)

Figure 3.6.1. The log plasma concentration-time profiles of indocyanine green alone ( +) and following administration of low (18.4 mg, o) and high (74 mg/kg, 0) doses of phenytoin in a rabbit (#15). The lines of best fit for the terminal elimination phases are shown. 83.

□ □ □ z 0 H ' t­ < ~ 1.B zw u z B.S 0u < l: (/) < -I a. B. 1 0 0.1 0.2 0.3 0.4 0.5 TIME CHRS)

Figure 3.6.2. The log plasma concentration-time profiles of indocyanine green alone ( +) and following administration of low (18.4 mg,o) and high (74 mg/kg,a) doses of phenytoin in a rabbit (#16). The lines of best fit for the terminal elimination phases are shown. 84.

~ 188 ...J :I: t!) 68 :J' 'J z 0 t-t I- < ~ 18 I-z w u z 6 lfl 0 0 (.) < :I: (/) < ...J a. 1.0 a 8. I 8.2 8.3 a ... a.s TIME CHRS)

Figure 3.6.3. The log plasma concentration-time profiles of indocyanine green alone ( +) and following administration of low (18.4 mg,o) and high (74 mg/kg,a) doses of phenytoin in a rabbit (#17). The lines of best fit for the terminal elimination phases are shown. 85.

TABLE 3.7.1

Patient Data and Concurrent Drug Therapy in Rapid Metabolizers of Phenytoin

Patient ~ Ag_e_ Concurrent Drug Therapy A.La M 42 carbamazepine J.L. M 57 carbamazepine, valproate, clonazepam I.D. M 25 sulthiame, methylphenobarbitone A.F.8 F 62 dexamethazone, carbamazepine J.c.a F 28 chlormethiazole, methylphenobarbitone J.s.a F 27 methylphenobarbitone

a alcoholic 86.

3.7 Rapid Metabolizers of Phenytoin

The patients in this study were selected from patients (Table 3.7.1) who were found to have sub-therapeutic plasma concentrations of phenytoin (Table 3.7.2) despite repeated phenytoin estimations and whose compliance to the prescribed dosage regimen was believed to be good.

There were 6 patients, who on admission to the study had trough plasma phenytoin concentrations ranging from 0 to 5 µg/ml despite dosage regimens ranging from 300 mg/day to 1.2 g/day. The half-lives of phenytoin after test doses of 300 mg of the drug ranged from 3.4 to 9.4 hours (mean=6.7 hr, s.d.= 2.1, Table 3.7.2). There were only a limited number of blood samples taken (4-5) and the plasma concentrations declined in a monoexponential fashion. Examples are shown in Figures 3.7.1 and 3.7.3.

Although 4 of the 6 patients were classified as alcoholics (Table 3.7.1) on the basis of their medical records, there was no alcohol detected in any of the samples assayed . This was probably due to the period of stay in hospital prior to the admission to the study.

The protein binding of phenytoin was also studied in 5 of these patients, it ranged from 12.8 to 14.8% free (Table 3.7.2). Plasma albumin concentrations were normal for patients I.D., J.C. 87.

TABLE 3.7.2

Pharmaeokinetie Data for Rapid Metabolizers of Phenytoin

Daily Plasma Plasma Phenytoin Cone.# Albumin 1.1.l.2. Free phenytoin Patient Dose* (mg) (µg/ml) Cone. (g/1) .(hrl 00

A.L. 300 0 5.2 12.8 J.L. 600 2.5 41 9.0 14.4 I.D. 700 3 47 7.0 13.6 A.F. 1100 5 3.4 J.C. 1200 2 36 8.2 14.8 J.S. 600 4 7.0 13.4 mean (s.d) 6.7 (2.1)

* Daily phenytoin dose on admission to the study. # Trough plasma phenytoin concentration at prescribed dosage. 88.

and J.L. ranging from 36 to 41 g/I (Table 3.7.2) while the other three did not have enough samples for albumin analysis.

Two subjects (A.L. and I.D.) with rapid elimination half­ lives of phenytoin were also dosed with a single dose of carbamazepine. A.L. who was on chronic carbamazepine therapy had a half-life of 15.5 hours (Fig. 3.7.2) whilst I.D., who had not previously taken carbamazepine, had a half-life of 13 hours. (Fig. 3.7.4)

One patient (G.A.) who was shown to have a normal terminal elimination half-life for phenytoin of 18 hours was found to have a short elimination half-life for carbamazepine of 7.6 hours (Table 3.7.3). 89.

TINE CtiRS)

Figure 3.7.1. Log plasma concentration-time profile of phenytoin following administration of a single 300 mg oral dose of phenytoin to the subject A.L. 90.

,.._ 10 ...J X: '....,

0 2 ◄ 6 8 10 12 1 ◄ 16 18 20 TIME CHRS)

Figure 3.7.2. Log plasma concentration-time profile of carbamazepine following administration of a single 400 mg oral dose of drug to the subject A.L. 91.

,a ◄ 8 12 16 28 24 28 32 TIME CHRS)

Figure 3.7.3. Log plasma concentration-time profile of phenytoin following administration of a single 300 mg oral dose of phenytoin to the subject I.D. 92.

0 6 10 16 20 25 30 35 TIME CHRS)

Figure 3.7.4. Log plasma concentration-time profile of carbamazepine following administration of a single 400 mg oral dose of drug to the subject I.D. 93.

TABLE 3.7.3

A Comparison of Phenytoin and Carbamazepine Half-lives

Patient ebeo~toio tll2 Carbamazepine lll2' Qarbamazepi ne (h r) (h r) Regimen A.L. 5 15.5 chronic I.D. 7 13 single G.A. 18 7.6 chronic 94.

3.8 The Specificity of Phenytoin Estimations by the EMIT Technique

The specificity of the E.M.I.T. technique for the determination of plasma phenytoin concentration was checked by comparison of the concentrations determined by this technique and by the specific G.L.C. method. Seventeen randomly selected plasma samples from the patients and 3 blank samples to which phenytoin had been added were assayed by the two methods. There was no significant difference between the values. The equation describing a plot of phenytoin estimations as measured by E.M.I.T. (y) against G.L.C.(x) was :

y = 0.552 + 0.987 X the correlation coefficient was 0.997 (Table 3.8.1 ).

Pooled rabbit plasma was also assayed for phenytoin by both the EMIT technique and by G.L.C. Excellent agreement between the two methods was obtained (Table 3.8.2).

It is known that there is some cross reactivity of the assay with HPPH. Consequently, it was decided to check if there was any significant inference of HPPH on the phenytoin determinations when measuring high phenytoin (and consequently high HPPH) concentrations. The interference was found to be small. An HPPH concentration of 20 µg/ml, which exceeds even the relatively high HPPH concentrations seen in rabbits, gave a result of only 1.5 µg/ml phenytoin equivalents (Table 3.8.3). Furthermore, a sample 95.

containing phenytoin at 96 µg/ml and HPPH at 100 µg/ml, gave a phenytoin result which was only 3% high. 96.

TABLE 3.8.1 A Comparison of Phenytoin Concentrations as Measured by EMIT and G.L.C.

Patient EMIT G.L.C. 1 3.0 2.8 2 1.6 1.5 3 2.5 2.5 4 7.8 7.0 5 1.9 1.7 6 3.2 3.4 7 15.2 13.9 8 13.6 13.5 9 18.5 17.8 10 10.7 10.6 1 1 11.0 11.0 12 35.0 34.0 13 39.0 37.0 14 46.0 48.0 15 53.0 56.0 16 20.0 20.0 17 42.7 39.0 18 Recovery 5.0 4.9 19 II 10.0 10.0 20 II 30.0 27.8 97.

TABLE 3.8.2

Comparison of Phenytoin Concentrations by G.L.C. and E.M.I.T. Assays in Pooled Rabbit Plasma

Rabbit No. G.L.C. E.M.I.T 5 7.8 7.8 6 12.8 12.0 8 72.0 70.8 9 56.2 57.2 10 41.8 42.2

Phenytoin concentration in µg/ml. 98.

TABLE 3.8.3

The Effect of HPPH on the Estimation of Phenytoin HPPH Cone. Phenytoin Equivalents Sample (µg/m I) (µg/m I)

1 1 0.2 2 5 0.6 3 10 1.0 4 20 1.5 5 30 2.1 6 40 2.5 7 50 3.4 8 80 5.1 9 100 5.6 99.

4. DISCUSSION

4.1 Phenytoin Overdosed Patients

The plasma elimination profiles for the 12 overdosed patients in this study were divided into 3 groups on the basis of whether they observed:-

1) Apparent first order elimination kinetics over the concentration range measured (Fig. 3.1.1 ).

2) Apparent zero order elimination kinetics over the concentration range measured (Fig. 3.1.2).

3) Apparent zero order or first order elimination kinetics but with an initial plateau phase (Fig. 3.1.3)

It is of note that the plateau phase was seen in 3 patients at plasma concentrations where only elimination phases were seen in other patients (groups 1,2).

4.1.1 Apparent First Order Elimination Kinetics

There were 3 patients (Fig. 3.1.1) in whom the elimination of phenytoin was first order. Half-lives calculated from this data were up to 106 hrs, which all greatly exceed the normal range of half-lives at therapeutic concentrations of 15-30 hrs (Section 1 .3.2) 100.

The time course of plasma concentrations of a drug eliminated by saturable mechanisms will appear log-linear if a short enough portion is taken in isolation. This proposition, however, is unlikely for the present data, since saturable kinetics should be apparent over the wide range of plasma concentrations observed. There are 2 possible reasons dose dependent elimination profiles were not observed in these patients.

Firstly, there may be a subset of the population for whom Km is a very large and consequently these subjects will have first order kinetics. Subjects have been described who have a familial defect in the enzymatic pathway which converts phenytoin to its para-hydroxylated metabolite, HPPH. These slow metabolizers of phenytoin were found to have half-lives ranging from 30 hours to 43 hours (Vasko fil...fil., 1979). Unfortunately, no time course of plasma concentrations were given and it is not clear whether the half-lives quoted are true half-lives based on first order elimination kinetics or estimated half-lives based on a limited range of plasma concentrations in a dose dependent profile. The first order kinetics observed in the 3 "slow" metabolizers may also be due to a lack of product inhibition (Ashley and Levy, 1972). Slow metabolizers of phenytoin may not allow sufficient accumulation of the inhibitory metabolite, which may itself be cleared relatively rapidly, to alter the pharmacokinetics of phenytoin elimination. However, the hypothesis that the non-linear kinetics of phenytoin is due to product has not been supported in recent years. 101.

Secondly, there may be a parallel "non-saturable" elimination pathway to that of the usual saturable pathway. This alternate process could become the dominant mode of elimination if the normal saturable pathway is deficient or absent. Thus, if V m for the saturable pathway is very low, the elimination of phenytoin should be close· to first order even at very high plasma concentrations (Arnold and Gerber, 1969).

There are two possibilities for the parallel non-saturable (first order) elimination of phenytoin. The non-saturable metabolism could involve the conversion phenytoin to its most important metabolite, HPPH, by a different enzyme than that responsible for its saturable metabolism; or there could be a non­ saturable pathway which does not involve the production of HPPH. Examples of both possibilities are known with other drugs. Ethanol is metabolised to acetaldehyde by two different pathways while the various metabolites of salicylic acid are produced by saturable and non-saturable pathways (Levy, 1971 ). For phenytoin, only the second possibility has been considered, but the literature is contradictory on whether a parallel pathway exists. Based on a review of the literature, Garrettson and Jusko (1974) concluded that the excretion of HPPH accounts for 70 to 90% of the dose of phenytoin, that this fraction is independent of the dose of phenytoin, and consequently, that a major metabolic pathway parallel to the formation HPPH does not exist. Unless the alternative pathway had nearly identical Michaelis-Menten parameters as for HPPH formation, the urinary 102.

Table 4.1.1 Michaelis-Menten Parameters for Phenytoin

Dose Rate Maximal Elimination Rates of Phenytoin Reference

( mg/kg/day) mg/kg/day btg/ml/day This Study 6.9 1 .2* 2.0 (Table 3.1.4) 4.5 2.9* 4.9 5.6 4.0* 6.6 7.1 5.2* 8.6 5.4 6.8* 11.4

5.3 - 8.4 Allen fil....fil., 1979 8.5 (2.0) (mean and SO) Jung tl_al., 1980

11.5* 19.1 Chaiken tl_al., 1979

6.5* 10.9 Atkinson & Shaw, 1973

* Volume of distribution assumed to be 0.6 I/kg. 103.

recovery of HPPH would not be a relatively constant fraction of the dose, but would decrease with increasing dosage.

However, in a study by Eadie et al. (1976) 3 of the 4 subjects given increasing doses of phenytoin, demonstrated an inverse relationship between increasing phenytoin dose and excretion of HPPH, thus suggesting that a parallel pathway does exist. Furthermore, Woodbury and Swinyard (1972) stated that at a dose of 100 mg, 60 to 85% of the phenytoin is excreted as HPPH, at a dose of 250 mg, 76% and at a dose of 500 mg, only 50% is excreted as HPPH, but no details or references were given in support of this data. However, a detailed review of the literature (Fig. 4.1.1) does not support this inverse relationship. This data suggests that the percentage of phenytoin excreted as HPPH is independent of the dose of phenytoin and that there is no parallel pathway of significance.

4.1.2 Apparent Saturable Kinetics

The patients who demonstrated zero order elimination had maximal elimination rates (1.2 to 6.8 mg/kg/day) which were similar to those previously determined Vmax values for low doses

(less than 300 mg) of phenytoin of 5.3 to 8.4 mg/kg/day (Table 4.1.1 ). However, a particularly interesting finding was that the Vmax values for 5 of the 6 subjects studied were less than their daily dose of phenytoin. This explains why these subjects had overdosed on their medication. The results do not indicate why there was 104.

excessive accumulation in the sixth subject, although it is possible that his dosage had been in larger than prescribed.

It is perhaps significant that the subject with the highest maximal elimination rate (6.8 mg/kg/day) for phenytoin in the present study was also being treated with carbamazepine. Carbamazepine is known to induce its own metabolism as well as that of phenytoin (Hansen et al., 1971) and other drugs (Warren et al., 1980). 105.

4.1.3 Plateau Phase Elimination Kinetics

An interesting aspect of the plasma concentration-time profiles was the plateau observed in 3 subjects (Fig. 3.1.3) following cessation of phenytoin therapy.

The plateau and/or continued rise in plasma phenytoin concentrations could be due to:- a) Continued absorption, and/or b) redistribution within body compartments.

Phenytoin is poorly soluble in the gastrointestinal tract and the plateau phase could be due to prolonged absorption (Section 1). Rises and plateaus of plasma phenytoin concentrations have been previously documented lasting 1 day (Gerber et al., 1972; Pruitt et al., 1975), 3 days (Gill et al., 1978) and 4 days (Matzke et al., 1979). In most cases, the phenomenon has been attributed to the effects of delayed absorption. The larger doses encountered in drug overdose will exacerbate this situation. A large drug mass may form with a subsequent reduction in the rate of absorption.

It is unlikely that the plateau is due to a limitation of the solubility of the drug in plasma since the maximum solubility in plasma is approximately 140 µg/ml (Section 1), well in excess of the plasma concentration (SO µg/ml) observed in overdosed patients. 106.

In one of the 3 subjects (W.M. Fig 3.1.3), the duration of the continued rise in plasma phenytoin concentration (7 days) exceeds that which could be reasonably explained on the basis of continuing absorption. In one previous report of a drug overdose in which phenytoin concentrations peaked at 24 hours, the author attributed the phenomenon to a "redistribution of phenytoin by unknown processes" (Pruit et al., 1975).

Recently, Gannaway and Mawer (1981) gradually increased phenytoin doses to the individual threshold of intoxication in 11 patients with poorly controlled epilepsy. One interesting observation was that the same daily dose of phenytoin tended to give higher serum drug concentrations after intoxication than before. They put forward the "tenable hypothesis" that phenytoin had accumulated in a slow compartment such as adipose tissue, and that the daily dose was effectively supplemented over several months by slow release from this depot.

The studies in the rabbit indicate that the plateau effect, need not be caused by prolonged absorption. This effect was reproduced in the rabbit after intravenous adminstration of high doses of phenytoin; thus one can conclude that the plateau or continued rise in plasma concentrations in the rabbit is not due to continued absorption. In view of this data, it was decided to investigate the possible relationship between phenytoin dose and its distribution in the rabbit. It was hypothesised that a significant relative change in distribution might contribute to the unusual 107.

plateaus and rises in plasma phenytoin concentrations observed in both the present and previous reports in man.

Extensive tissue distribution of the drug occurred for both high and low doses. However, it was the results for fat which were of particular interest, as fat was the only tissue which showed a relative change in phenytoin distribution on going from low to high dose. For the low dose, there was a relatively constant proportion (5.5%) in fat (Table 3.3.2), whereas for the high dose, phenytoin in fat accounted for 10-20% of the dose (Table 3.3.2).

The distribution of phenytoin preferentially into fat tissues following a high dose suggests that redistribution of phenytoin from the fat compartment back into plasma may be the factor which most contributes to the plateau or continued rise in plasma concentrations observed in the rabbits.

Two cases of fatal phenytoin poisoning have been described in which tissue levels of phenytoin were measured (Laubscher; 1966, Tichner and Enselberg; 1951 ). Phenytoin was shown to be concentrated in brain, liver, kidney and muscles, but no fat levels were reported. The relative concentrations of phenytoin in tissue to plasma (g/g) were: brain 1.7, liver 6.0, kidney 2.5, (Laubscher; 1966). These are not in good agreement with our animal data, although this could be attributed to the different times after dosing that distribution was studied. The human data was for distribution at death, 4 days after the overdose, at which time plasma 108.

concentrations had fallen from 94 to 45 µg/ml. The rabbit data was for 2 hours after the (I.V.) dose.

4.2 Protein Binding

Protein binding of phenytoin was examined in order to determine the possible contribution of this parameter to the rising or plateau concentrations observed in the overdosed patients and rabbits. In the case of drug overdose, it is more likely that there would be a significant change in the free fraction of drug as the drug concentration in plasma approaches or even may exceed the protein concentration (as is the case for salicylate within its therapeutic range). In such a situation, there will be a redistribution of the free drug with a resulting increase in the apparent volume of distribution of the drug.

If the protein binding of phenytoin was saturable, the disproportionate increase in the free phenytoin concentration with increasing dose would accentuate the substrate saturation effect that phenytoin exerts on its metabolizing enzymes. Any other factors which lead to a displacement of phenytoin from its binding site could also contribute to this effect. In this respect it was of interest that HPPH was found not to displace phenytoin (10 µg/ml) from albumin (Table 3.4.2) over the HPPH concentration range 1 to 100 µg/ml. This result confirms a previous observation that phenytoin appears to bind to albumin about threefold more tightly 109.

than HPPH (Woodbury and Glasko; 1972) and thus was not likely to be displaced by this metabolite.

In the present studies, however, it was found that free phenytoin fractions determined on specimens collected from the patients when first admitted were not significantly different from those observed when plasma concentrations had returned to the therapeutic range (Table 3.4.1 ). This finding confirms similar observations of Hooper et al., (1973). It thus appears unlikely that interpatient differences in protein binding was a significant factor in producing the three different patterns· of plasma concentrations.

In normal individuals the binding of phenytoin to plasma proteins, principally albumin, varies between 85 and 92% (Lunde et al., 1970) at therapeutic plasma phenytoin concentrations. Earlier studies had suggested that intersubject variation in the degree of protein binding was considerable (Hooper et al., 1974, Booker and Darcey; 1973). However, these data have been criticized on methodological grounds (Barth et al., 1976). More recent results indicate that in normal adults and otherwise healthy epileptics, the variability in protein binding is usually no more than twofold (Monks et al., 1978, Barth et al., 1976, Richens et al., 1983), although this still represents a clinically significant variation.

Albumin concentrations may affect protein binding. It has been shown that for every 0.1 g/100 ml decrease in albumin concentration that there is an absolute increase in the unbound 110.

phenytoin fraction of 1% (Perucca; 1980). However, since phenytoin is a low clearance drug in man, a reduction in plasma protein binding capacity alone would be expected to result in a fall in total drug concentration at steady state, without any change in concentration of free, pharmacologically active drug. Albumin determinations on several overdosed patients (Table 3.4.2) showed that they were all within the normal range for plasma albumin concentrations (36 g/1 to 47 g/1).

These data· suggest that changes in protein binding are not significant following phenytoin overdose. This is in agreement with the finding in a single patient, who following a phenytoin overdose, had a plasma concentration of 87 µg/ml, a spinal fluid concentration of 5.8 µg/ml and salivary concentration between 4 and 6 µg/ml (Kutt et al., 1964). These data would indicate that the proportion of free phenytoin in this patient was between 5 and 7%. However, there is a report that in a patient with a total phenytoin plasma concentration of 96.5 µg/ml the "percentage free phenytoin was 2.5 times that observed at therapeutic concentrations" (Chaiken et al., 1979). The reason for this different finding is not clear. There may be methodological problems in determining free phenytoin concentrations. It was recently demonstrated for instance that if serum rather than plasma is used with Worthington filters then the normal free proportion of phenytoin is 25% rather than the expected 10%. Temperature is also known to significantly alter protein binding of phenytoin (Lunde et al., 1970; Hooper et al., 1973). For 111.

this reason all protein binding experiments in the present studies were carried out at 370c by equilibrium dialysis.

The effect of two commonly used anticoagulants and the effect of freezing and thawing on plasma samples on the protein binding of phenytoin were also investigated. There was no significant variations in protein binding of phenytoin in heparinized or EDTA tubes as compared with plain clotted tubes (Table 3.4.4). However, heparin has been shown to increase the free fraction of diazepam, propranolol and lignocaine (Desmond et al., 1980) and consequently all samples were collected into EDTA tubes.

There was only a relatively small difference observed in the binding of phenytoin to plasma proteins following freezing and rethawing.

Contrary to the finding in human plasma, the proportion of free phenytoin in rabbit plasma increased from approximately 8 to 12% over the concentration range of 5 to 100 µg/ml. A Scatchard plot of these data (Figure 3.4.1) indicated that there is one binding site for phenytoin on each albumin molecule with a dissociation constant of 74.9 umol.l (Ka = 13.4 1/mol). It is difficult to compare these results with those for human plasma. In the present work, the change in binding in human plasma was not sufficient to allow accurate determinations of the Scatchard parameters. Rudman et al., (1971) reported that there is one class of phenytoin binding site for phenytoin in human albumin although again the range of 1 1 2 .

concentrations was limited by the solubility of phenytoin in plasma and, consequently was too narrow to permit accurate calculations of the number of binding sites or the dissociation constant. However, it has been reported that phenytoin does bind to only one site with an apparent association constant of 1.86 X 104 1/mol (Monks et al., 1978).

The in vitro results (Table 3.4.5) for the binding of phenytoin in rabbit plasma were confirmed by the in viva data. There was a higher unbound fraction of phenytoin in plasma taken from rabbits in the high dose distribution study than from plasma of rabbits from the low dose distribution study (Table 3.4.6). Furthermore, data for a single rabbit given both a low dose and a high dose of phenytoin confirmed these observations.

It is concluded that in the rabbit, protein binding is somewhat concentration dependent. Consequently, the increase in free fraction with increasing phenytoin concentration may partly contribute to the effect of dose on tissue distribution observed in this model. In this respect, the rabbit is not an ideal model for studying the events which occur in man following an overdose of phenytoin. 1 1 3 .

4.3 The Effect of Phenytoin on Blood Pressure and Hepatic Blood El.Q.W.

It was considered that two parameters which might contribute to the plateau in the elimination of phenytoin from both rabbits and man were blood pressure and liver blood flow. Compromised hepatic perfusion would be expected to decrease drug clearance, at least for a high clearance drug, while a substantial fall in blood pressure might alter tissue perfusion and consequently affect drug distribution.

The rate of elimination of ICG has been shown to be highly dependent on hepatic blood flow in man (Caesar et al., 1961; Weigand et al., 1960). This is consistent with the findings that ICG has a high extraction ratio and is cleared only by the liver with no enteroheptic circulation (Caesar et al., 1961 ). Consequently, it has been used for estimating liver blood flow in man and has also been demonstrated to be applicable to studying blood flow in the dog (Wheeler et al., 1958), the rat (Caesar et al., 1961) and more recently the rabbit (Heintz et al., 1986).

An increase in ICG clearance indicates an increase in hepatic blood flow. The data showed (Table 3.6.1) that, assuming an extraction ratio for ICG of 0.7 (Caesar et al., 1961 ), the mean liver blood flow increased from 24 ml/min to 38 ml/min following the low dose of phenytoin. This was an interesting, but unexplained 114.

observation, as there is no supporting data in the literature to indicate that low doses of phenytoin increase liver perfusion.

The clearance for a low dose of phenytoin in the rabbit was near liver blood flow. Thus phenytoin is a high clearance drug in the rabbit. This contrasts with the situation in man where phenytoin is a low clearance drug. Large doses of phenytoin decreased liver perfusion in this situation. Hepatic blood flow was estimated to have fallen slightly from 24 ml/min to 19.1 ml/min. This effect would thus be expected to also cause a small fall in the clearance of phenytoin. This decrease in hepatic blood flow is consistent with the hypotension observed secondary to a decrease in cardiac output. A cardiac depressant effect of large doses of phenytoin is not unexpected.

These findings about the effect of phenytoin on hepatic blood flow refers only to the time period over which the clearance of ICG was studied, from 4 to 4.3 hours after the infusion of phenytoin. At this time period, the blood pressure was still depressed following the high dose of phenytoin, but changes in hepatic may have been more marked at earlier times after the infusion.

Recently, it has been recognised that the spectrophotometric assay for indocyanine green used in the present study is not entirely reliable (Heintz et al., 1986). Clearance may have been underestimated because an impurity in the ICG is also 115.

measured by the spectrophotometric assay. The impurity is eliminated more slowly than ICG in this species leading to an overestimation of ICG concentrations at later times. However, the results for ICG clearance in the present study are similar to the data obtained by Heintz et al., (1986) and the error should not be sufficient to alter the general conclusions reached above.

The plateau phase kinetics and the decrease in liver perfusion was also associated with a prolonged hypotensive effect. This data indicated that there were probably changes in tissue perfusion as well which may have also contributed to the plateau phase.

Changes in the blood pressure in the rabbit prompted an examination of the literature to see whether a similar effect had been reported to occur in man following phenytoin overdose.

The characteristics of phenytoin elimination evident in these data indicate that the rabbit was not a perfect model for the study of phenytoin overdose in man. Apart from the differences in the effects of large doses of phenytoin on blood pressure and the finding that phenytoin is a high clearance drug in the rabbit, there was the previous observation that there is some non-linearity in protein binding in rabbit plasma not observed in human plasma. However, despite these limitations, the comparative studies in rabbits did illustrate that prolonged absorption is not the only possible explanation for the plateau phase observed in man. The 1 1 6 .

plateau may be related to distributional phenomena, although again a contribution from prolonged absorption is still likely.

4.4 Rapid Metabolizers of Phenytoin

Large doses of phenytoin may not always result in toxic plasma concentrations. Some patients were shown to require large doses of phenytoin for adequate control of their epilepsy.

The normal elimination half-life for phenytoin is said to be between 15 and 30 hours. Slow metabolizers of phenytoin have been described and this has been shown to be a familial characteristic in some cases (Kutt et al., 1964) However, the capacity to metabolise drugs rapidly has generally been associated with induction of enzymatic activity by an exogenous substance rather than with an inherited trait. A familial ability to metabolise drugs rapidly has not been reported.

A number of drugs have been shown to enhance the clearance of phenytoin. Folate has been shown to decrease steady state concentrations of phenytoin (Baylis et al., 1971) while dichloralphenazine was found to double the clearance of phenytoin (Riddell et al., 1980). Kater et al. (1969) reported that chronic alcoholics had a shorter half-life than normal (Hansen et al., 1971) and showed that patients given carbamazepine for more than 9 days had a mean half-life which decreased significantly (p < 0.001) from 1 1 7 .

10.6 to 6.4 hours. The latter data is unusual in that the mean half­ life before carbamazepine is already very short.

The data obtained in the present investigation confirms some of these previous reports. Of the 6 subjects studied, 4 were alcoholics on the basis of previous medical histories. However, no alcohol was detected in any plasma samples on the day of the investigation (Table 3.7.1). This may be because the subjects were already in-patients of the hospital with no ready access to alcohol. One of the non-alcoholic subjects (J.l.) was concurrently receiving carbamazepine, as were 2 of the alcoholic group (A.L., A.F.) (Table 4.7.2) Short phenytoin half-lives in these subjects may be attributed to the effect of carbamazepine.

The one remaining non-alcoholic subject was being treated with sulthiame and prominal. It has been found that after administration of phenobarbitone, epileptics metabolize phenytoin more rapidly, although this is of little clinical significance in the majority of patients (Buchanan & Sholiton; 1972, Kutt et al., 1969).

Sulthiame has been commonly prescribed with phenytoin. (LaVeck et al., 1962; Liske et al., 1963; Liu; 1966) Contrary to earlier belief, sulthiame has little intrinsic anticonvulsant activity (Gordon, 1964). When used in conjunction with phenytoin it apparently elevates plasma phenytoin levels by inhibiting the metabolism of phenytoin (Hansen et al., 1968; Olesen and Jensen, 118.

1969). For this subject (I.D.) then, the short phenytoin half-life is not so readily explained on the basis of enzyme induction.

The protein binding of phenytoin in each of the subjects studied was within the expected normal limits as were the albumin concentrations (Table 4.7.3). Consequently, the rapid clearance of phentyoin in these subjects can not be attributed to some perturbation of the free fraction of phenytoin.

As a group, the rapid elimination half-life for phenytoin can generally be explained as being the result of enzyme induced metabolism by either carbamazepine, phenobarbitone or alcohol. This explanation does not as readily apply to the subject I.D. With the effects of phenobarbitone on the elimination of phenytoin remaining equivocal and sulthiame being expected to prolong the elimination of phenytoin, the reason for the 7 .0 hour half-life is unclear. The present data is insufficient to support the hypothesis that there are congenitally rapid metabolizers of phenytoin.

The data demonstrates that although most patients are controlled by normal dosage regimes of approximately 300 mg/day, some patients may require over 1 g/day and/or more frequent dosage intervals for adequate control. Such subjects may be more suitably controlled by an anticonvulsant with a longer elimination half-life. However, this raises the question as to whether patients who metabolize phenytoin rapidly also metabolize other anticonvulsants rapidly. 119.

Some data relevant to this question were obtained for the relationship between phenytoin and carbamazepine metabolism. Phenytoin and carbamazepine are both oxidatively metabolized as their major routes of elimination. However, for phenytoin this involves para-hydroxylation of the phenyl ring while for carbamazepine, formation of the 10, 11-epoxide is the major metabolic pathway. It might be concluded from this data that there would not be any correlation between the metabolic capacity for phenytoin and carbamazepine for an individual. However, it was shown recently that non-metabolisers of· sparteine were poor hydroxylators of debrisoquine and extensive hydroxylators of debrisoquine were extensive sparteine metabolisers. This correlation was found despite their different oxidative pathways of metabolism; sparteine undergoes N-oxidation followed by dehydration while debrisoquine is metabolized primarily by apiphatic 4-hydroxylation and to a minor degree by aromatic hydroxylation.

The interrelationship between drug metabolic capacities is further confused by the finding that metabolism of antipyrine, a primary marker for drug metabolic activity, showed no correlation with either debrisoquine or sparteine metabolism. Furthermore, Idle et al. (1976) found that the para-hydroxylation of phenytoin was impaired in the poor debrisoquine metaboliser phenotype. 120.

In the present work, the patient A.L. who was shown to be a rapid metaboliser of phenytoin had a normal elimination half-life for carbamazepine, while the patient G.A. who had a normal phenytoin half-life, rapidly metabolised carbamazepine. A third patient (1.0.), was found to be a rapid metaboliser of both phenytoin and carbamazepine (Table 3.7.4). This limited data shows that there is no clear correlation between the capacities for phenytoin and carbamazepine metabolism.

There are significant therapeutic implications of such data. The poor or slow metaboliser phenotype is susceptible to drug accumulation with related drug toxicity at normal dosage regimes. At the same standard dosage the rapid or extensive metaboliser phenotype will be poorly controlled as sub-optimal plasma concentrations will be attained. Furthermore, for drugs with reactive or toxic metabolites, the rapid metaboliser phenotype may also be at risk of developing drug toxicity besides having sub­ optimal control.

4.5 The Reliability of Phenytoin Estimations by the Enzyme Immunoassay Technigue (E.M.I.T)

The reliability of the E.M.I.T. technique for the determination of plasma phenytoin concentrations has been previously investigated. (Bastiani et al., 1973, Rubenstein et al., 1972) 121.

There was a significant correlation between E.M.I.T. and G.L.C. The results indicated that the G.L.C. assay was consistently giving lower estimations which may suggest that G.L.C. is more specific than E.M.I.T. although this difference was not significant.

The effect of HPPH on the estimation of phenytoin was found to be relatively small despite HPPH concentrations up to 100 µg/ml, a concentration that would unlikely be attained even ifollowing an overdose of phenytoin.

Phenytoin concentrations in pooled rabbit plasma obtained using either EMIT or G.L.C. were in good agreement indicating that there was nothing in rabbit plasma that interfered with the E.M. I.T. determinations. 122.

5. CONCLUSIONS

i) Overdose may lead to saturation of the normal processes of absorption, distribution and excretion.. Consequently, extrapolation of data obtained following .admin~tration of therapeutic doses of phenytoin may be misleading if used to predict the 1fffte course of toxicity following an overdose. 'tune

ii) Highly variable profiles may occur following phenytoin overdose. These vary from linear to non-linear elimination. Initially, plasma concentrations may not decline and may even rise for many days.

iii) Redistribution of phenytoin within body compartments together with continuing absorption of this poorly soluble drug may be important factors contributing to the unusual profiles observed.

iv) Saturation of protein binding following phenytoin overdose is unlikely to occur.

v) Some saturation of plasma protein binding occurs in the rabbit and this may partly contribute to the disproportionate distribution of phenytoin into fat which was observed when large doses of phenytoin were given. 123.

vi) On the basis of rabbit data, it may be reasonable to conclude that phenytoin distributes significantly into fat particularly following overdose. Slow redistribution from the fat back into plasma may be one of the reasons that plasma phenytoin concentrations plateau and/or increase over several days.

vii} Some epileptic patients may require large doses of phenytoin for adequate control. Short half-lives in these subjects may be attributed to enzyme induction by concommitantly administered drugs. The possibility that some people may be congenitally rapid metabolisers of phenytoin has not been either proved or disproved.

ix) Rapid metabolizers of phenytoin are not necessarily rapid metabolisers of other anticonvulsants. Consequently, such patients may be more easily controlled by treatment with a more slowly eliminated anticonvulsant such as carbamazepine. 124.

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7.1 Assay for HPPH.

The concentration-response relationship for HPPH was found to be linear over the concentration range of 10 to 100 µg/ml for standards extracted from spiked rabbit plasma (Figure 7.1 ). The lower sensitivity limit of the assay was 1.0 µg/ml and the assay was found to have a day-to-day coefficient of variation of 5.0%.

7 .2 Assay for Phenytoin

The concentration versus change in absorbance for plasma standards over the concentration range of 0.5 to 15µg/ml was linear when plotted on the modified logit function paper (Figure 7.2). Samples which had concentrations which exceeded the upper standard were diluted appropriately to fall within the range of the standards. The day-to-day reproducibility of the system in our hands was good with a coefficient of variation of 5.3%. I ?>1

-0 u; 3 iii ...C G) C i- 2 0. 0. ::c 0 :;::: 1 al a:

0 20 40 60 80 100 120 HPPH Concentration (µg/ml)

Figure 7 .1. Typical standard curve used to measure the concentration of HPPH. The y-axis is the ratio of the peak heights of HPPH to internal standard (p-tolylphenytoin) measured by GLC. I ~8

() FOR Emrt• FREELEVEL.. ANALYSES. USE TWO, USE THIS FORM ONLY WITH REAGENT CYCLE SEMI-LOGARITHMIC GRAPH PAPER. OR USE LOTS OF N01 (e.g., N01A. N018, elC.J Concen1ra11on (,ag/ml) MODEL 11 WITH NO CONSTANTS ON THE CP-5000. 2 0 2.5 30 40 5.0 60 7.0 B 0 9.0 10.0 150 200 250 180

f---- 1- ·+-i-r -, ' , 170 170 f--- -+- =t=r-=+-~:-t!--r-lt-, -t-1 -• ++ 160 ------+ :--;++ -=- .. -~-~--- 160 t===t'·=- =-=-=-tf-=-=-=-=-=-=-=-tt-=-=-=-=-=-=1l-=-=-=-=-=-=--=--=--=-=--=--=--=--=-=--=--=--=-:-=--=--=1-,- I~

.---- H--r- _j_'-~ BO>----+---~- I..-.., 80 -f----~ ·r+ - '-t- ---r--:--r 70 -- 70 1..-'.' Emit:!l 60 PHENYTOIN {s~a) ASSAY 50

LOT N01 DATE OPER ,o 2.0 2.5 3.0 4.0 5 0 6.0 7 0 8.0 9.0 10.0 15 0 a =0.146011 b=0.523944 Model 11 Concenua11on (.1-1giml) <£ Syn 1983. 68172-15

Figure 7.2. A typical standard curve for the assay of phenytoin in plasma using the EMIT technique. Changes in absorbance are plotted on the standard EMIT modified logit function paper.