Epilepsia. 2037-45, 1979.
Raven Press, New York
Estimation of Plasma Unbound Phenobarbital Concentration by Using Mixed Saliva *Kazuyo Nishihara, *Katsuyoshi Uchino, "Yukiya Saitoh, TYutaka Honda, *Fuji0 Nakagawa, and "Zenzo Tamura *Hospital Pharmacy, Faculty of Medicine, University of Tokyo, and
t Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan
Summary: Relationships among plasma total (C,), plasma protein unbound (Cf) and mixed salivary (C,) concentrations of phenobarbital (PB) were studied in 29 epileptic patients. A highly significant correlation was observed between C, and Cf. Although a significant correlation was observed between Cf and C,, C,/Cfratio was only 0.63. When C, was corrected by using pKa (7.41) and pH of saliva to estimate free PB concentration (C,,, 3, a highly significant correlation was obtained between Cf and C,, f r and C,,, r/Cf ratio was 0.96. The temporal course of C,, obtained from C, after an oral single dose of 50 mg PB in a healthy adult male volunteer was approximated as a triexponential equation. The peak'time was 2 hr after ingestion and apparent half-life was 66 hr. The prediction of minimum concentrations at steady state on the basis of these parameters corresponded well with actually obtained C,,, following repetitive administration of PB.
c
Determination of the blood level of antiepileptic drugs has now acquired sufficient credit to be considered clinically useful in the treatment of epilepsy. To avoid toxic effects due to overdoses and to maintain effective therapeutic concentration, frequent determinations of the blood level of antiepileptic drugs and careful evaluation of the individual differences in the pharmacokinetic parameters concerning absorption, distribution, metabolism, and excretion of the drug are necessary. Most antiepileptic drugs, especially phenytoin and phenobarbital (PB), are present in plasma, partly bound to protein and partly in the
free (protein unbound) form. Both clinical efficacy and toxic effects of antiepileptic drugs are generally considered to be closely related to the concentration of the drugs in their free form in plasma, rather than their total drug concentration in plasma. It has also been pointed out that the concentration of antiepileptic drugs in the cerebrospinal fluid is almost equal to their free drug concentration in plasma (Lund et al., 1972; Troupin and Friel, 1975). Therefore determination of the free form of antiepileptic drugs is clinically very important as the indicator of active drug concentrations within the central nervous system ~
Received April 10, 1978. Key words: Phenobarbital-Salivary tion-Pharmacokinetics.
~~~
concentration-Plasma total concentration-Plasma Free Concentra-
37
38
K . NISHIHARA ET AL.
(Booker and Darcey, 1973). However, the determination of plasma free concentration of antiepileptic drugs requires larger amounts of blood and time-consuming complicated procedures of ultrafiitration or equilibrium dialysis prior to the determination procedure, and, therefore, is unsuitable as a routine clinical laboratory testing. In recent years salivary concentration of phenytoin has been reported to be almost identical to its free concentration in plasma (Bochner et al., 1974). Clinically, the use of saliva has several advantages: frequent samplings may be taken without syringes and a hospital setting, and there need be no worry about time and place for sample collection (Danhof and Breimer, 1978). In contrast to phenytoin, the concentration of PB in saliva is not equal to plasma free PB concentration (Cook. et al., 1975; Schmidt and Kupferberg, 1975; Troupin and Friel, 1975; Homing et al., 1977). If plasma free PB concentration can be estimated accurately from saliva PB concentration, it should be of great clinical significance. It has been pointed out that the transfer of PB from plasma to saliva is influenced by the pH of saliva at the time.of saliva secretion (Schmidt and Kupferberg, 1975; Troupin and Friel, 1975; Homing et al., 1977). This is explained by the closeness of pKa, an electrolytic dissociation exponent, of PB (7.41) to the pH of plasma (7.40). Matin et al. (1974) applied the equation of Jacobs (1940) to estimate plasma free concentration of tolbutamide from the concentration in saliva by using pKa values. Dvorchik and Vesell (1976) examined the validity of the Matin’s equation in estimating the free fraction of various drugs, including PB, in plasma. They used data of free fractions obtained by equilibrium dialysis in the literatures, assumed saliva pH as 6.5, and concluded that the calculated estimation of free fraction agreed with the dialyzed free fraction in plasma. We employed a similar theoretical equation to estimate plasma free PB concentrations
Epilepsia, Vol. 20, February 1979
from saliva PB concentrations in epileptic patients, and we obtained satisfactory results. In addition, pharmacokinetic parameters of PB were calculated on the basis of serially obtained saliva PB concentrations in a healthy male volunteer, and they were examined against the theoretically. predicted steady state PB levels. MATERIALS AND METHODS
Twenty-nine epileptic outpatients (16 males with an age range of 9-70 years, and 13 females with an age range of 11-40 years) at the Department of Neuropsychiatry of Tokyo University Hospital and Fujisawa Hospital and a healthy male volunteer were the subjects of this study. The patients were given PB and/or primidone with or without phenytoin one to three times a day%gularly for at least 1 month prior to the determination. Five milliliters of blood was collected into a heparinized polypropylene syringe and about 2-4 ml of mixed saliva was simultaneously collected with mouth and tongue movement alone into a polypropylene centrifuge tube with a stopper. The salivary pH was determined as soon as possible by a pH meter (TOA Electronic Ltd., Model HM-SB, Tokyo, Japan), and the saliva samples were refrigerated until determination. Plasma was separated from blood sample and also refrigerated until determination. One of the authors, a 46-year-old healthy male with a body weight of 62 kg, ingested a single oral dose of 50 mg of PB’, and serial collections of mixed saliva were made every 1-2 hr for 14 hr and with extended intervals for 48 hr. After 1 month of the experiment, the volunteer again ingested PB tablets, 50 mg twice a day regularly for 12 days at 10 a.m. and at 10 p.m. Mixed I PB tablets containing 50 mg of PB in 6 tablets, prepared by the Hospital Pharmacy, University of Te kyo.
ESTIMATION OF FREE PB BY USING SALIVA saliva samples were collected just before 10 a.m. on the 8th, loth, and 12th days. Mixed saliva samples were centrifuged at 3,000 rpm by a centrifugal separator (Kubota KC-70, Tokyo, Japan) for 5 min and 0.4-4 ml of the supernatant was used for PB determination; 0.4 ml of plasma was used for total PB determination. According to the method of Tanimura et al. (1976), 2 ml of plasma was pipetted into curved seamless cellulose tubing (Visking Co., Type 20/32, 130 mm length); both open ends of the tubing were held tightly by a silicone rubber stopper against the inner wall of the glass vessel. The glass vessel holding the tubing inside was centrifuged at 3,000 rpm by a centrifugal separator (Hitachi 20 PR-5, Tokyo, Japan) for 40 min. Two hundred microliters of ultrafiltrate was used for the determination of free PB concentration in plasma. Ultraviolet spectrophotometric method (Saitoh et al., 1973) was used for the determination of PB concentration. The equation of Ma,tin et al. (1974) was employed: 1 + IO(PHP - PKa) (Eq. 1) f' = cs 1 + 10'pHs - pKa) where Cf is free PB concentration in plasma, C, is saliva PB concentration, pHp is pH of plasma, pHs is pH of saliva, and pKa is pKa of PB. When pHp was assumed as 7.40 and pKa of PB was 7.41 (Kakemi et al., 1963, the following equation was obtained to calculate the estimated free PB concentration (C-, f) from saliva concentration (C& Cestf = 1.977C,/( 1
+ lopHS-
7.41)
(Eq. 2)
Table 1. As shown in Fig. la, a statistically significant correlation (p < 0.005) was found between plasma total (CJ and plasma free (Cf) PB concentrations. The Ct/Ct ratio was 0.53 2 0.01 (mean 2 ME). Although a significant correlation (p < 0.005) was found between free PB concentrations in plasma and salivary PB concentrations (C,), a considerable discrepancy between values of both concentrations was observed, and C,/Cf ratio was only 0.63 & 0.02, as shown in Fig. lb. On the other hand, the calculated C,,, values showed a very good correlation with actually determined plasma free PBfoncentrations (Cf). As indicated in Fig. lc, the correlation coefficient was 0.938, and Cest$C,ratio was 0.96 & 0.03, indicating an almost 1:l relationship. Table 2 shows reliable correlations between Cf and Ce, f at varying pH and C, in 3 patients given varying amounts of PB. Significant correlations (p < 0.005)were observed between daily PB dose (mg/kg * day) and plasma total PB concentration (Fig. 2a), between PB dose and plasma free PB concentration (Fig. 2b), and between PB dose and estimated free PB concentration (Fig. 2c), in epileptic patients receiving PB with or without phenytoin. The PB concentrations of mixed saliva samples collected serially after a single oral administration of PB (0.81 mg/kg) were determined, and estimated free PB concentrations were plotted logarithmically against time as illustrated in Fig. 3. The estimated free PB concentrations showed a maximum value about 2 hr after ingestion followed by TABLE 1. The range and means of phenobarbital (PB) concentrations observed
RESULTS
The range of pH of 54 mixed saliva samples obtained from 29 patients was 5.4-7.6, with an average pH of 6.74 & 0.06 (mean & ME). The ranges and means of plasma total, plasma free, and saliva, as well as estimated free PB concentrations, are given in
39
Range
PB concentration CP Cf CP C, P (I
(pg/ml) .
6.2-42.2 3.1-23.0 1.4-13.3 3.4-23.1
Mean 2 ME (pg/ml) 18.7 2 1.21 9.7 f 0.62 6.0 t 0.38 9.3 0.63
*
See text.
Epilepsia. Vol. 20, February 1979
20
30 46
Total PB concn. in plasma (pg/ml)
10
1-a
io
I
15
.
20 25
i n saliva (w/ml)
10
PB concn.
5
1-b ..
.
1-c
J
5 10 15 20 25 Estimated free PB concn. (pg/ml)
I
FIG. 1. Relationship between a. plasma total and plasma free phenobarbital (PB) concentrations, b. salivary and plasma free PB concentrations, and c. estimated free and plasma free PB concentrations in 29 epileptic patients receiving PB andor primidone (n = 54). a. Plasma total PB-plasma free PB concentration: y = 0 . 4 9 9 ~+ 0.406, r = 0.964, p < 0.005. b. Salivary PB-plasma free PB concentration: y = 1.446~+ 1.118, r = 0.872, p < 0.005. c. Estimated free PB-plasma tkee PB concentration: y = 0 . 9 3 0 ~+ 1.106, r = 0.938, p < 0.005.
I
ESTIMATION OF FREE PB BY USING SALIVA
41
TABLE 2. Comparison between Cf and C,,, calculated from C, and pH of mixed saliva in 3 patients
H.H.
54
M
K.O.
37
F
H.Y.
36
F
6.3 6.4 TO 6.6 7.2
7.4 7.0 4.4 5.6 6.5
13.8 12.7 6.3 9.7 8.0
14.5 13.7 8.4 10.8 7.6
6.2 7.0
12.3 7.5
23.1 10.7
23.0 10.8
" See text.
a rapid decrease, then a slow decline after 8 and CJCf ratio was 0.63 +- 0.02. It has been hr. The curve was approximated by Eq. (3): pointed out in the previous reports (Schmidt and Kupferberg, 1975; Troupin and Friel, 1975; Homing et al., 1977) that transfer of PB from plasma to saliva was where t is time after PB administration. The affected by the change of salivary pH. In apparent half-life of the tail phase of the order to bring the C$Cf ratio closer to a unit, correction of C, is necessary. curve was 66 hr. McAuliffe et al. (1977) reported that there Theoretical prediction of the minimum was a significant linear relationship bevalues of PB at steady state [Cpredf]min was tween the logarithm of the ratio of free PB made by superimposing Eq. (3), and the concentration in plasma to salivary PB concalculation was approximated by the folcentration and salivary pH. They only lowing equation: briefly commented on pKa and Matin's [C,,, f]min = 5.0(1 - e-0.12m)(Eq. 4) equation, but they did not use it. Instead, they recommended an empirical equation where n is the number of repeated adminis- which was valid for a pH range of 6.3-7.8. tration. The predicted values at 10 a.m. on If the equation of Matin et al. (1974) is the 8th, IOth, and 12th days by using Eq. (4) applicable to the PB transfer, the relationwere in good agreement with the values of ship between the logarithm of the ratio of estimated free PB concentrations calcu- free plasma PB to PB in saliva and salivary lated from actually determined saliva PB pH of wider range should lead to a sigconcentrations (Table 3). moid curve. Furthermore, when we applied our saliva data on the equation of McAuDISCUSSION liffe, the obtained approximated free Several reports have been made on the PB concentration/Cf was 0.91 f 0.03 relationship between salivary and plasma (mean f ME), and the relationship beconcentrations of PB. The C,/C, ratios re- tween approximated free PB and plasma ported were 0.29-0.32 (Cook et al., 1975; free PB was y = 0 . 8 4 2 ~+ 2.203 (r = Schmidt and Kupferberg, 1975; Homing et 0.900,~< 0.005). al., 1977), and C,/Cf ratio was 0.60 (Troupin In our present investigation, estimation and Friel, 1'175). In the present investiga- of free PB concentration in plasma was tion performed on 54 paired samples of made theoretically from salivary PB conplasma and saliva obtained from 29 epilep- centration by using salivary pH and Eq. (2). tic patients, C,/C1 ratio was 0.33 f 0.31, The C,,, p/Cf ratio was 0.96 2 0.03, which Epilepsia, Vol. 20, February 1979
W
.
8
2-a
d
I
20
0-
0
c
6
.
10
a 15 c -d
rl
fd
2
m
1 2 3 4 PB dose (mg/kg-day)
9
40-
1 2 3 4 PB dose (mg/kg day)
I
I
I
1 2 3 4 PB dose (mg/kg day)
2-c
FIG.2. Relationship between a. dose and plasma total concentration of phenobarbital (PB),b. dose and plasma free concentration of PB,and c. dose and estimated free concentration of PB,in 18epileptic patients with an age range of 11-70 years receiving PB (n = 29) a. Dose-plasma total concentration ofPB: y = 8.515~+ 3.252, r = 0.864, p < 0.005. b. Dose-plasma free concentration of PB:y = 4.11 lx + 2.348, r = 0.757, p < 0.005. c. Dose-estimated free concentration of PB: y = 4.222~+ 1.643, r = 0.797, p < 0.005.
E-c
0
a
d
fd
2 tn
ESTIMATION OF FREE PB BY USING SALIVA 1.0
43
'I
w
1
4
d
8
12 16 20 24 28 32 36 40 44 48 Time after administration(h0ur) FIG. 3. Temporal course of estimated free phenobarbital (PB) concentrations (C-, in a normal subject given a single oral dose of PB 50 mg (0.806 mg/kg). The range of C. was 0.22-0.49 p g / d and that of C, was 0.42-0.92 & n l . The solid line represents approximation by the following triexponential equation:
,
was almost close to a unit. The Cest/Cf ratio dose of PB and plasma PB concentration did not show significant difference when we used pKa value of 7.2 (Waddell and Butler, 1957) where the relationship between Cest and CfW ~ Sy = 1.129~- 0.46, r =. 0.924, and pKa value of 7.3 (Bush and SandersBush, 1972) where the relationship was = 1.048~- 0.243, r = 0.931. &die et al. (1977) recently reported that the relationship between administered daily JJ
TABLE 3. Comparison between the minimum values of the estimated free phenobarbital (PB) concentrations ([C, &,,), which were calculated from actually determined saliva PB concentrations after repeated administration, and the predicted minimum PB concentrations ([C,, &,3obtained by using Eq. (4). Days of repeated administration PB concentration
8
10
12
can be better approximated by a curve rather than a linear approximation. They assumed saturation of metabolic process of PB for their reasoning. On the basis of data obtained in our present investigation, as illustrated in Fig. 2, it can be concluded that each of the plasma total, free, and estimated free PB concentrations was linearly proportionate to the administered PB dose (0.63-3.80 mg/kg.day) and curve fitting is not necessary2. Previous information on the pharmacokinetic aspects of PB in human body indicated that plasma total PB concentration peaked at about 2 hr after oral administration, and the apparent half-life was 41 hr (Johanessen and Strandjord, 1975). Viswanathan et al. (1978) reported that This conclusion between daily dose and steady state PB concentrations requires circumspection, since most of the epileptic patients in this study were receiving both PB and phenytoin, and the inhibitory effect of phenytoin (Morselli et al., 1971) on PB elimination was not taken into account. Epilepsia, Vol. 20, February 1979
K. NISHIHARA ET AL.
44
mean peak time of serum PB was 2 hr after an oral dose of 30 mg PB in 5 normal male subjects. As shown in Fig. 3, accurate information on the peak time of PB was obtained by using saliva. The finding that the temporal course of PB concentrations was approximated by triexponential functions also confirms the usefulness of salivary PB determination and the validity of a twocompartment model for the pharmacokinetic analysis of PB in the human body. By using Eq. (4), minimum free PB concentration in the steady state after repeated administration of the same amount of PB (50 mg twice a day at 12 hr intervals, 1.61 mg/kg day) was calculated on the basis of single dose experiment. The pharmacokinetic parameters in Eq. (3) were assumed to be constant throughout the period of repeated administrations. The predicted minimum value of free PB concentrations after repeated administration by using Eq. (4) showed good agreement with the estimated values calculated from actually determined saliva PB concentration by using Eq. (2). According to prediction, 10, 12, and 19 days were required to reach 90,94, and 99%, respectively, of the steady state PB concentrations; this finding supported the view of Schmidt (1977) that 2-3 weeks of repeated administrations were required before steady state was reached. The collection of mixed saliva can easily be made in various situations, such as immediately after clinical seizures, late at night, or early in the morning, at home or at working places, and at desired intervals, and the use of syringes and immediate centrifugation is not necessary. In addition, the determination procedure of saliva PB concentration is much simpler and less time consuming than the one for plasma free PB concentration, which requires a delicate technique of ultrafiltration. Therefore the former can be widely used as a routine clinical laboratory testing. The high reliability of estimating free PB concentration by applying theoretical equation on saliva PB ‘
Epilepsia, Vol. 20, February 1979
concentration has great significance in everyday clinical practice because of the ease of frequent saliva collection, and the advantage of direct monitoring of substantially effective PB concentrations in the body fluid. It can be concluded that pharmacokinetic analysis of PB and theoretical dosage regimens designed for individual patients can be made accurately and easily by using concentrations of PB in the mixed saliva. REFERENCES Bochner F, Hooper WD,Sutherland JM, Eadie MJ, and Tyrer JH. Diphenylhydantoin concentrations in saliva. Arch Neurol 31:57-59, 1974. Booker HE and Darcey B. Serum concentrations of free diphenylhydantoin and their relationship to clinical intoxication. Epilepsia 14: 177- 184, 1973. Bush MT and Sanders-Bush E. Phenobarbital, mephobarbital, and metharbital, and their metabolites, chemistry and methods for determination. In: DM Woodbury, JK Penry, and RP Schmidt (Eds), Antiepileptic Drugs, Raven Press, New York, p 295, 1912. Cook CE, Amersen E, Poole WK, Lesser P, and O’Tuama L. Phenytoin and phenobarbital concentrations in saliva and plasma measured by radioimmunoassay. Clin Pharmacol Ther 18:742-741, 1975.
Danhof M and Breimer DD. Therapeutic drug monitoring in saliva. Clin Pharmacokinet 3:39-57, 1978. Dvorchick BH and Vessel1 ES. Pharmacokinetic interpretation of data gathered during therapeutic drug monitoring. Clin Chem 22:868-878, 1976. Eadie MJ, Lander CM, Hooper WD and Tyrer JH. Factors influencing plasma phenobarbitone levels in epileptic patients. Br J Clin Pharmacol 4:541-547, 1977.
Homing MG, Brown L, Nowlin J, Lertratanangkoon K, Kellaway P, and Zion TE. Use of saliva in therapeutic drug monitoring. Clin Chem 23: 157- 164, 1977.
Jacobs MH. Some aspects of cell permeability to weak electrolytes. Cold Spring Harbor Symp Quant Biol 8:30-39, 1940.
Jdhannessen SI and Strandjord RE. Absorption and protein binding in serum of several anti-epileptic drugs. In: H Schneider, D Janz, C GardnerThorpe, H Meinardi, and AL Sherwin (Eds), Clinical Pharmacology of Anti-Epileptic Drugs, Springer-Verlag, New York, 1975, p 268. Kakemi K, Arita T, Hori R, and Konishi R. Absorption and excretion of drugs. XXX. Absorption of barbituric acid derivatives from rat stomach. Chem Pharm Bull 15: 1534- 1539, 1967. Lund L, Berlin A, and Lunde RKM. Plasma protein binding of diphenylhydantoin in patients with epilepsy. Agreement between the unbound fraction
ESTIMATION OF FREE PB BY USING SALIVA
45
in plasma and the concentration in the cerebrospi- aprks ]'ingestion et la demi-vie apparente est de 66 nal fluid. Clin Pharmacol Ther 13: 196-200, 1972. heures. La prevision des concentrations minimum a Matin SB, Wan SH, and Karam JH. Pharmacokinetics l'etat d'equilibre sur la base de ces parametres correof tolbutamide: Prediction by concentration in spond bien au C,& effectivement obtenu apr& administration rkpttee de PB. saliva. Clin Pharmacol Ther 1 6 1053- 1058, 1974. McAuliffe JJ, Sherwin AL, Leppik IE, Fayle SA, and (J .-L. Gastaut, Marseilles ) Pippenger CE. Salivary levels of anticonvulsants: A practical approach to drug monitoring. Neurol- RESUMEN Ogy 27:409-413, 1977. En 29 pacientes epilepticos se han estudiado las reMorselli PL, Rizzo M,and Garattini S. Interaction between phenobarbital and diphenylhydantoin in laciones entre la concentraci6n de fenobarbital (PB) animals and in epileptic patients. Ann N YAcadSci total en plasma (C,), de PB no ligado a las proteinas plasmaticas (Cf), y de PB en saliva mixta (C,). A pesar 17988-107, 1971. Saitoh Y, Nishihara K, Nakagawa F and Suzuki T. de que se observ6 una correlaci6n significativa entre Improved microdetermination for diphenylhydan- Cf y C,, el cociente C,/Cf fue solo de 0.63. Cuando se toin in blood by UV spectrophotometry. J Pharm comgib C, utilizando pKa (7.41) y el pH de la saliva para calcular la concentracidn de PB libre (CeSt&,se Sci 62:206-210, 1973. Schmidt D. Die Behandlung der Epilepien mit Hilfe obtuvo una correlaci6n altamente significativaentre Cf der Blutspiegelbestimmung von Antiepileptika. y C,,, f , y el cociente Ces, r/C, fue de O.%. El curso temporal de Ces, obtenido de C, tras una dosis Onica Nervenarzt 48: 183- 196, 1977. Schmidt D and Kupferberg HJ. Diphenylhydantoin, de 50 mg de PB en un voluntario adulto sano fuC apphenobarbital, and primidone in saliva, plasma and roximado como una ecuaci6n triexponencial. El punto mas alto se consiguid a las 2 horas de la ingestidn y la cerebrospinal fluid. Epilepsia 16:735-741, 1975. Tanimura Y , Kimura M , Ohkawa Y , Saitoh Y, vida media aparente fue de 66 horas. La prediccidn de Nakagawa F, and Suzuki T. In vitro determination las concentraciones minimas en el "steady state," of drug-protein binding by hemodialysis. %th An- basandose en estos padmetros, correspondib bien nual Meeting of Pharmaceutical Society of Japan, con la C,,, obtenida tras la adminestracidn de PB. Nagoya, Japan, April 1976. (A. Portera Sanchez, Madrid) Troupin AS and Friel P. Anticonvulsant level in saliva, serum, and cerebrospinal fluid. Epilepsia l6:223 -227, 1975. Viswanathan. CT, Booker HE, and Welling PG. Bioavailability of oral and intramuscular ZUSAMMENFASSUNG phenobarbital. J Clin Pharmacol 18: 100- 105, Bei 29 epileptischen Patienten wurde die Konzentra1978. tion en des Phenobarbital (PB) und ihre gegenseitigen Waddell WJ and Butler TC. The distribution and Beziehungen im Gesamtplasma (C,), im plasexcretion of phenobarbital. J Clin Invest maeiweiaungebundenen (Cf)und gemischten Speichel 361217-1226, 1957. (C,) untersucht. Eine hoch signifikante Korrelation wurde zwischen C, und Cr gefunden. Obwohl eine signifikante Beziehung zwischen Cf und C, gefunden R~SUME wurde, betrug das CJCrVerhaItnis nur 0.63. Wenn C, Les relations entre les concentrations de phenobar- korrigiert wurde durch pka (7.41) und das pH des bital (PB) dans le plasma total (C,), dans les proteines Speichels, um die freie PB-Konzentration (C,& f) zu libres du plasma (Cf), et dans les secretions salivaires bestimmen, wurde eine statistisch hochsignifikante mixtes (C,) sont ktudikes chez 29 sujets kpileptiques. Korrelation zwischen Cr und Ces, erreicht; Das Une correlation hautement significative est observee C,,, ,/CrVerhiiltnis war 0.96. Der zeitliche Verlauf von entre C, et C,. Bien qu'une correlation significative soit C,,, aus Cs nach einer einzigen oralen Dosis von SO observkq entre Cf et C,, le rapport C,/Cf n'est que de mg PB bei gesunden mannlichen freiwilligen Erwach0.63. Lorsque C, est comge par l'utilisation de pKa senen wurde als triexponentielle Gleichung geschatzt. (7.41) et du pH de la salive pour apprkcier la concen- Das Maximum wurde 2 Stunden nach Einnahme tration de PB libre (C,,, f), une correlation hautement erreicht und die Halbwertzeit betrug 66 Stunden. significative est obtenue entre Cf et C,,, et le rapport Die Vorhersage der minimalen Konzentration bei C,,, r/Cfest de O.%. L'evolution dans le temps de C,,, einem steady state auf der Basis dieser Parameter obtenu & partir de C,, a p r h une prise orale unique de entspricht sehr wohl den aktuell erzielten C,,, r 50 mg de PB par un volontaire adulte de sexe masculin Werten aufgrund wiederholter Verabfolgung von PB. se prksente approximativement comme une equation (D. Scheffner, Heidelberg) triexponentielle. Le pic maximum se situe 2 heures
Epilepsia, Vol. 20, February 1979
Raven Press, New York
Estimation of Plasma Unbound Phenobarbital Concentration by Using Mixed Saliva *Kazuyo Nishihara, *Katsuyoshi Uchino, "Yukiya Saitoh, TYutaka Honda, *Fuji0 Nakagawa, and "Zenzo Tamura *Hospital Pharmacy, Faculty of Medicine, University of Tokyo, and
t Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan
Summary: Relationships among plasma total (C,), plasma protein unbound (Cf) and mixed salivary (C,) concentrations of phenobarbital (PB) were studied in 29 epileptic patients. A highly significant correlation was observed between C, and Cf. Although a significant correlation was observed between Cf and C,, C,/Cfratio was only 0.63. When C, was corrected by using pKa (7.41) and pH of saliva to estimate free PB concentration (C,,, 3, a highly significant correlation was obtained between Cf and C,, f r and C,,, r/Cf ratio was 0.96. The temporal course of C,, obtained from C, after an oral single dose of 50 mg PB in a healthy adult male volunteer was approximated as a triexponential equation. The peak'time was 2 hr after ingestion and apparent half-life was 66 hr. The prediction of minimum concentrations at steady state on the basis of these parameters corresponded well with actually obtained C,,, following repetitive administration of PB.
c
Determination of the blood level of antiepileptic drugs has now acquired sufficient credit to be considered clinically useful in the treatment of epilepsy. To avoid toxic effects due to overdoses and to maintain effective therapeutic concentration, frequent determinations of the blood level of antiepileptic drugs and careful evaluation of the individual differences in the pharmacokinetic parameters concerning absorption, distribution, metabolism, and excretion of the drug are necessary. Most antiepileptic drugs, especially phenytoin and phenobarbital (PB), are present in plasma, partly bound to protein and partly in the
free (protein unbound) form. Both clinical efficacy and toxic effects of antiepileptic drugs are generally considered to be closely related to the concentration of the drugs in their free form in plasma, rather than their total drug concentration in plasma. It has also been pointed out that the concentration of antiepileptic drugs in the cerebrospinal fluid is almost equal to their free drug concentration in plasma (Lund et al., 1972; Troupin and Friel, 1975). Therefore determination of the free form of antiepileptic drugs is clinically very important as the indicator of active drug concentrations within the central nervous system ~
Received April 10, 1978. Key words: Phenobarbital-Salivary tion-Pharmacokinetics.
~~~
concentration-Plasma total concentration-Plasma Free Concentra-
37
38
K . NISHIHARA ET AL.
(Booker and Darcey, 1973). However, the determination of plasma free concentration of antiepileptic drugs requires larger amounts of blood and time-consuming complicated procedures of ultrafiitration or equilibrium dialysis prior to the determination procedure, and, therefore, is unsuitable as a routine clinical laboratory testing. In recent years salivary concentration of phenytoin has been reported to be almost identical to its free concentration in plasma (Bochner et al., 1974). Clinically, the use of saliva has several advantages: frequent samplings may be taken without syringes and a hospital setting, and there need be no worry about time and place for sample collection (Danhof and Breimer, 1978). In contrast to phenytoin, the concentration of PB in saliva is not equal to plasma free PB concentration (Cook. et al., 1975; Schmidt and Kupferberg, 1975; Troupin and Friel, 1975; Homing et al., 1977). If plasma free PB concentration can be estimated accurately from saliva PB concentration, it should be of great clinical significance. It has been pointed out that the transfer of PB from plasma to saliva is influenced by the pH of saliva at the time.of saliva secretion (Schmidt and Kupferberg, 1975; Troupin and Friel, 1975; Homing et al., 1977). This is explained by the closeness of pKa, an electrolytic dissociation exponent, of PB (7.41) to the pH of plasma (7.40). Matin et al. (1974) applied the equation of Jacobs (1940) to estimate plasma free concentration of tolbutamide from the concentration in saliva by using pKa values. Dvorchik and Vesell (1976) examined the validity of the Matin’s equation in estimating the free fraction of various drugs, including PB, in plasma. They used data of free fractions obtained by equilibrium dialysis in the literatures, assumed saliva pH as 6.5, and concluded that the calculated estimation of free fraction agreed with the dialyzed free fraction in plasma. We employed a similar theoretical equation to estimate plasma free PB concentrations
Epilepsia, Vol. 20, February 1979
from saliva PB concentrations in epileptic patients, and we obtained satisfactory results. In addition, pharmacokinetic parameters of PB were calculated on the basis of serially obtained saliva PB concentrations in a healthy male volunteer, and they were examined against the theoretically. predicted steady state PB levels. MATERIALS AND METHODS
Twenty-nine epileptic outpatients (16 males with an age range of 9-70 years, and 13 females with an age range of 11-40 years) at the Department of Neuropsychiatry of Tokyo University Hospital and Fujisawa Hospital and a healthy male volunteer were the subjects of this study. The patients were given PB and/or primidone with or without phenytoin one to three times a day%gularly for at least 1 month prior to the determination. Five milliliters of blood was collected into a heparinized polypropylene syringe and about 2-4 ml of mixed saliva was simultaneously collected with mouth and tongue movement alone into a polypropylene centrifuge tube with a stopper. The salivary pH was determined as soon as possible by a pH meter (TOA Electronic Ltd., Model HM-SB, Tokyo, Japan), and the saliva samples were refrigerated until determination. Plasma was separated from blood sample and also refrigerated until determination. One of the authors, a 46-year-old healthy male with a body weight of 62 kg, ingested a single oral dose of 50 mg of PB’, and serial collections of mixed saliva were made every 1-2 hr for 14 hr and with extended intervals for 48 hr. After 1 month of the experiment, the volunteer again ingested PB tablets, 50 mg twice a day regularly for 12 days at 10 a.m. and at 10 p.m. Mixed I PB tablets containing 50 mg of PB in 6 tablets, prepared by the Hospital Pharmacy, University of Te kyo.
ESTIMATION OF FREE PB BY USING SALIVA saliva samples were collected just before 10 a.m. on the 8th, loth, and 12th days. Mixed saliva samples were centrifuged at 3,000 rpm by a centrifugal separator (Kubota KC-70, Tokyo, Japan) for 5 min and 0.4-4 ml of the supernatant was used for PB determination; 0.4 ml of plasma was used for total PB determination. According to the method of Tanimura et al. (1976), 2 ml of plasma was pipetted into curved seamless cellulose tubing (Visking Co., Type 20/32, 130 mm length); both open ends of the tubing were held tightly by a silicone rubber stopper against the inner wall of the glass vessel. The glass vessel holding the tubing inside was centrifuged at 3,000 rpm by a centrifugal separator (Hitachi 20 PR-5, Tokyo, Japan) for 40 min. Two hundred microliters of ultrafiltrate was used for the determination of free PB concentration in plasma. Ultraviolet spectrophotometric method (Saitoh et al., 1973) was used for the determination of PB concentration. The equation of Ma,tin et al. (1974) was employed: 1 + IO(PHP - PKa) (Eq. 1) f' = cs 1 + 10'pHs - pKa) where Cf is free PB concentration in plasma, C, is saliva PB concentration, pHp is pH of plasma, pHs is pH of saliva, and pKa is pKa of PB. When pHp was assumed as 7.40 and pKa of PB was 7.41 (Kakemi et al., 1963, the following equation was obtained to calculate the estimated free PB concentration (C-, f) from saliva concentration (C& Cestf = 1.977C,/( 1
+ lopHS-
7.41)
(Eq. 2)
Table 1. As shown in Fig. la, a statistically significant correlation (p < 0.005) was found between plasma total (CJ and plasma free (Cf) PB concentrations. The Ct/Ct ratio was 0.53 2 0.01 (mean 2 ME). Although a significant correlation (p < 0.005) was found between free PB concentrations in plasma and salivary PB concentrations (C,), a considerable discrepancy between values of both concentrations was observed, and C,/Cf ratio was only 0.63 & 0.02, as shown in Fig. lb. On the other hand, the calculated C,,, values showed a very good correlation with actually determined plasma free PBfoncentrations (Cf). As indicated in Fig. lc, the correlation coefficient was 0.938, and Cest$C,ratio was 0.96 & 0.03, indicating an almost 1:l relationship. Table 2 shows reliable correlations between Cf and Ce, f at varying pH and C, in 3 patients given varying amounts of PB. Significant correlations (p < 0.005)were observed between daily PB dose (mg/kg * day) and plasma total PB concentration (Fig. 2a), between PB dose and plasma free PB concentration (Fig. 2b), and between PB dose and estimated free PB concentration (Fig. 2c), in epileptic patients receiving PB with or without phenytoin. The PB concentrations of mixed saliva samples collected serially after a single oral administration of PB (0.81 mg/kg) were determined, and estimated free PB concentrations were plotted logarithmically against time as illustrated in Fig. 3. The estimated free PB concentrations showed a maximum value about 2 hr after ingestion followed by TABLE 1. The range and means of phenobarbital (PB) concentrations observed
RESULTS
The range of pH of 54 mixed saliva samples obtained from 29 patients was 5.4-7.6, with an average pH of 6.74 & 0.06 (mean & ME). The ranges and means of plasma total, plasma free, and saliva, as well as estimated free PB concentrations, are given in
39
Range
PB concentration CP Cf CP C, P (I
(pg/ml) .
6.2-42.2 3.1-23.0 1.4-13.3 3.4-23.1
Mean 2 ME (pg/ml) 18.7 2 1.21 9.7 f 0.62 6.0 t 0.38 9.3 0.63
*
See text.
Epilepsia. Vol. 20, February 1979
20
30 46
Total PB concn. in plasma (pg/ml)
10
1-a
io
I
15
.
20 25
i n saliva (w/ml)
10
PB concn.
5
1-b ..
.
1-c
J
5 10 15 20 25 Estimated free PB concn. (pg/ml)
I
FIG. 1. Relationship between a. plasma total and plasma free phenobarbital (PB) concentrations, b. salivary and plasma free PB concentrations, and c. estimated free and plasma free PB concentrations in 29 epileptic patients receiving PB andor primidone (n = 54). a. Plasma total PB-plasma free PB concentration: y = 0 . 4 9 9 ~+ 0.406, r = 0.964, p < 0.005. b. Salivary PB-plasma free PB concentration: y = 1.446~+ 1.118, r = 0.872, p < 0.005. c. Estimated free PB-plasma tkee PB concentration: y = 0 . 9 3 0 ~+ 1.106, r = 0.938, p < 0.005.
I
ESTIMATION OF FREE PB BY USING SALIVA
41
TABLE 2. Comparison between Cf and C,,, calculated from C, and pH of mixed saliva in 3 patients
H.H.
54
M
K.O.
37
F
H.Y.
36
F
6.3 6.4 TO 6.6 7.2
7.4 7.0 4.4 5.6 6.5
13.8 12.7 6.3 9.7 8.0
14.5 13.7 8.4 10.8 7.6
6.2 7.0
12.3 7.5
23.1 10.7
23.0 10.8
" See text.
a rapid decrease, then a slow decline after 8 and CJCf ratio was 0.63 +- 0.02. It has been hr. The curve was approximated by Eq. (3): pointed out in the previous reports (Schmidt and Kupferberg, 1975; Troupin and Friel, 1975; Homing et al., 1977) that transfer of PB from plasma to saliva was where t is time after PB administration. The affected by the change of salivary pH. In apparent half-life of the tail phase of the order to bring the C$Cf ratio closer to a unit, correction of C, is necessary. curve was 66 hr. McAuliffe et al. (1977) reported that there Theoretical prediction of the minimum was a significant linear relationship bevalues of PB at steady state [Cpredf]min was tween the logarithm of the ratio of free PB made by superimposing Eq. (3), and the concentration in plasma to salivary PB concalculation was approximated by the folcentration and salivary pH. They only lowing equation: briefly commented on pKa and Matin's [C,,, f]min = 5.0(1 - e-0.12m)(Eq. 4) equation, but they did not use it. Instead, they recommended an empirical equation where n is the number of repeated adminis- which was valid for a pH range of 6.3-7.8. tration. The predicted values at 10 a.m. on If the equation of Matin et al. (1974) is the 8th, IOth, and 12th days by using Eq. (4) applicable to the PB transfer, the relationwere in good agreement with the values of ship between the logarithm of the ratio of estimated free PB concentrations calcu- free plasma PB to PB in saliva and salivary lated from actually determined saliva PB pH of wider range should lead to a sigconcentrations (Table 3). moid curve. Furthermore, when we applied our saliva data on the equation of McAuDISCUSSION liffe, the obtained approximated free Several reports have been made on the PB concentration/Cf was 0.91 f 0.03 relationship between salivary and plasma (mean f ME), and the relationship beconcentrations of PB. The C,/C, ratios re- tween approximated free PB and plasma ported were 0.29-0.32 (Cook et al., 1975; free PB was y = 0 . 8 4 2 ~+ 2.203 (r = Schmidt and Kupferberg, 1975; Homing et 0.900,~< 0.005). al., 1977), and C,/Cf ratio was 0.60 (Troupin In our present investigation, estimation and Friel, 1'175). In the present investiga- of free PB concentration in plasma was tion performed on 54 paired samples of made theoretically from salivary PB conplasma and saliva obtained from 29 epilep- centration by using salivary pH and Eq. (2). tic patients, C,/C1 ratio was 0.33 f 0.31, The C,,, p/Cf ratio was 0.96 2 0.03, which Epilepsia, Vol. 20, February 1979
W
.
8
2-a
d
I
20
0-
0
c
6
.
10
a 15 c -d
rl
fd
2
m
1 2 3 4 PB dose (mg/kg-day)
9
40-
1 2 3 4 PB dose (mg/kg day)
I
I
I
1 2 3 4 PB dose (mg/kg day)
2-c
FIG.2. Relationship between a. dose and plasma total concentration of phenobarbital (PB),b. dose and plasma free concentration of PB,and c. dose and estimated free concentration of PB,in 18epileptic patients with an age range of 11-70 years receiving PB (n = 29) a. Dose-plasma total concentration ofPB: y = 8.515~+ 3.252, r = 0.864, p < 0.005. b. Dose-plasma free concentration of PB:y = 4.11 lx + 2.348, r = 0.757, p < 0.005. c. Dose-estimated free concentration of PB: y = 4.222~+ 1.643, r = 0.797, p < 0.005.
E-c
0
a
d
fd
2 tn
ESTIMATION OF FREE PB BY USING SALIVA 1.0
43
'I
w
1
4
d
8
12 16 20 24 28 32 36 40 44 48 Time after administration(h0ur) FIG. 3. Temporal course of estimated free phenobarbital (PB) concentrations (C-, in a normal subject given a single oral dose of PB 50 mg (0.806 mg/kg). The range of C. was 0.22-0.49 p g / d and that of C, was 0.42-0.92 & n l . The solid line represents approximation by the following triexponential equation:
,
was almost close to a unit. The Cest/Cf ratio dose of PB and plasma PB concentration did not show significant difference when we used pKa value of 7.2 (Waddell and Butler, 1957) where the relationship between Cest and CfW ~ Sy = 1.129~- 0.46, r =. 0.924, and pKa value of 7.3 (Bush and SandersBush, 1972) where the relationship was = 1.048~- 0.243, r = 0.931. &die et al. (1977) recently reported that the relationship between administered daily JJ
TABLE 3. Comparison between the minimum values of the estimated free phenobarbital (PB) concentrations ([C, &,,), which were calculated from actually determined saliva PB concentrations after repeated administration, and the predicted minimum PB concentrations ([C,, &,3obtained by using Eq. (4). Days of repeated administration PB concentration
8
10
12
can be better approximated by a curve rather than a linear approximation. They assumed saturation of metabolic process of PB for their reasoning. On the basis of data obtained in our present investigation, as illustrated in Fig. 2, it can be concluded that each of the plasma total, free, and estimated free PB concentrations was linearly proportionate to the administered PB dose (0.63-3.80 mg/kg.day) and curve fitting is not necessary2. Previous information on the pharmacokinetic aspects of PB in human body indicated that plasma total PB concentration peaked at about 2 hr after oral administration, and the apparent half-life was 41 hr (Johanessen and Strandjord, 1975). Viswanathan et al. (1978) reported that This conclusion between daily dose and steady state PB concentrations requires circumspection, since most of the epileptic patients in this study were receiving both PB and phenytoin, and the inhibitory effect of phenytoin (Morselli et al., 1971) on PB elimination was not taken into account. Epilepsia, Vol. 20, February 1979
K. NISHIHARA ET AL.
44
mean peak time of serum PB was 2 hr after an oral dose of 30 mg PB in 5 normal male subjects. As shown in Fig. 3, accurate information on the peak time of PB was obtained by using saliva. The finding that the temporal course of PB concentrations was approximated by triexponential functions also confirms the usefulness of salivary PB determination and the validity of a twocompartment model for the pharmacokinetic analysis of PB in the human body. By using Eq. (4), minimum free PB concentration in the steady state after repeated administration of the same amount of PB (50 mg twice a day at 12 hr intervals, 1.61 mg/kg day) was calculated on the basis of single dose experiment. The pharmacokinetic parameters in Eq. (3) were assumed to be constant throughout the period of repeated administrations. The predicted minimum value of free PB concentrations after repeated administration by using Eq. (4) showed good agreement with the estimated values calculated from actually determined saliva PB concentration by using Eq. (2). According to prediction, 10, 12, and 19 days were required to reach 90,94, and 99%, respectively, of the steady state PB concentrations; this finding supported the view of Schmidt (1977) that 2-3 weeks of repeated administrations were required before steady state was reached. The collection of mixed saliva can easily be made in various situations, such as immediately after clinical seizures, late at night, or early in the morning, at home or at working places, and at desired intervals, and the use of syringes and immediate centrifugation is not necessary. In addition, the determination procedure of saliva PB concentration is much simpler and less time consuming than the one for plasma free PB concentration, which requires a delicate technique of ultrafiltration. Therefore the former can be widely used as a routine clinical laboratory testing. The high reliability of estimating free PB concentration by applying theoretical equation on saliva PB ‘
Epilepsia, Vol. 20, February 1979
concentration has great significance in everyday clinical practice because of the ease of frequent saliva collection, and the advantage of direct monitoring of substantially effective PB concentrations in the body fluid. It can be concluded that pharmacokinetic analysis of PB and theoretical dosage regimens designed for individual patients can be made accurately and easily by using concentrations of PB in the mixed saliva. REFERENCES Bochner F, Hooper WD,Sutherland JM, Eadie MJ, and Tyrer JH. Diphenylhydantoin concentrations in saliva. Arch Neurol 31:57-59, 1974. Booker HE and Darcey B. Serum concentrations of free diphenylhydantoin and their relationship to clinical intoxication. Epilepsia 14: 177- 184, 1973. Bush MT and Sanders-Bush E. Phenobarbital, mephobarbital, and metharbital, and their metabolites, chemistry and methods for determination. In: DM Woodbury, JK Penry, and RP Schmidt (Eds), Antiepileptic Drugs, Raven Press, New York, p 295, 1912. Cook CE, Amersen E, Poole WK, Lesser P, and O’Tuama L. Phenytoin and phenobarbital concentrations in saliva and plasma measured by radioimmunoassay. Clin Pharmacol Ther 18:742-741, 1975.
Danhof M and Breimer DD. Therapeutic drug monitoring in saliva. Clin Pharmacokinet 3:39-57, 1978. Dvorchick BH and Vessel1 ES. Pharmacokinetic interpretation of data gathered during therapeutic drug monitoring. Clin Chem 22:868-878, 1976. Eadie MJ, Lander CM, Hooper WD and Tyrer JH. Factors influencing plasma phenobarbitone levels in epileptic patients. Br J Clin Pharmacol 4:541-547, 1977.
Homing MG, Brown L, Nowlin J, Lertratanangkoon K, Kellaway P, and Zion TE. Use of saliva in therapeutic drug monitoring. Clin Chem 23: 157- 164, 1977.
Jacobs MH. Some aspects of cell permeability to weak electrolytes. Cold Spring Harbor Symp Quant Biol 8:30-39, 1940.
Jdhannessen SI and Strandjord RE. Absorption and protein binding in serum of several anti-epileptic drugs. In: H Schneider, D Janz, C GardnerThorpe, H Meinardi, and AL Sherwin (Eds), Clinical Pharmacology of Anti-Epileptic Drugs, Springer-Verlag, New York, 1975, p 268. Kakemi K, Arita T, Hori R, and Konishi R. Absorption and excretion of drugs. XXX. Absorption of barbituric acid derivatives from rat stomach. Chem Pharm Bull 15: 1534- 1539, 1967. Lund L, Berlin A, and Lunde RKM. Plasma protein binding of diphenylhydantoin in patients with epilepsy. Agreement between the unbound fraction
ESTIMATION OF FREE PB BY USING SALIVA
45
in plasma and the concentration in the cerebrospi- aprks ]'ingestion et la demi-vie apparente est de 66 nal fluid. Clin Pharmacol Ther 13: 196-200, 1972. heures. La prevision des concentrations minimum a Matin SB, Wan SH, and Karam JH. Pharmacokinetics l'etat d'equilibre sur la base de ces parametres correof tolbutamide: Prediction by concentration in spond bien au C,& effectivement obtenu apr& administration rkpttee de PB. saliva. Clin Pharmacol Ther 1 6 1053- 1058, 1974. McAuliffe JJ, Sherwin AL, Leppik IE, Fayle SA, and (J .-L. Gastaut, Marseilles ) Pippenger CE. Salivary levels of anticonvulsants: A practical approach to drug monitoring. Neurol- RESUMEN Ogy 27:409-413, 1977. En 29 pacientes epilepticos se han estudiado las reMorselli PL, Rizzo M,and Garattini S. Interaction between phenobarbital and diphenylhydantoin in laciones entre la concentraci6n de fenobarbital (PB) animals and in epileptic patients. Ann N YAcadSci total en plasma (C,), de PB no ligado a las proteinas plasmaticas (Cf), y de PB en saliva mixta (C,). A pesar 17988-107, 1971. Saitoh Y, Nishihara K, Nakagawa F and Suzuki T. de que se observ6 una correlaci6n significativa entre Improved microdetermination for diphenylhydan- Cf y C,, el cociente C,/Cf fue solo de 0.63. Cuando se toin in blood by UV spectrophotometry. J Pharm comgib C, utilizando pKa (7.41) y el pH de la saliva para calcular la concentracidn de PB libre (CeSt&,se Sci 62:206-210, 1973. Schmidt D. Die Behandlung der Epilepien mit Hilfe obtuvo una correlaci6n altamente significativaentre Cf der Blutspiegelbestimmung von Antiepileptika. y C,,, f , y el cociente Ces, r/C, fue de O.%. El curso temporal de Ces, obtenido de C, tras una dosis Onica Nervenarzt 48: 183- 196, 1977. Schmidt D and Kupferberg HJ. Diphenylhydantoin, de 50 mg de PB en un voluntario adulto sano fuC apphenobarbital, and primidone in saliva, plasma and roximado como una ecuaci6n triexponencial. El punto mas alto se consiguid a las 2 horas de la ingestidn y la cerebrospinal fluid. Epilepsia 16:735-741, 1975. Tanimura Y , Kimura M , Ohkawa Y , Saitoh Y, vida media aparente fue de 66 horas. La prediccidn de Nakagawa F, and Suzuki T. In vitro determination las concentraciones minimas en el "steady state," of drug-protein binding by hemodialysis. %th An- basandose en estos padmetros, correspondib bien nual Meeting of Pharmaceutical Society of Japan, con la C,,, obtenida tras la adminestracidn de PB. Nagoya, Japan, April 1976. (A. Portera Sanchez, Madrid) Troupin AS and Friel P. Anticonvulsant level in saliva, serum, and cerebrospinal fluid. Epilepsia l6:223 -227, 1975. Viswanathan. CT, Booker HE, and Welling PG. Bioavailability of oral and intramuscular ZUSAMMENFASSUNG phenobarbital. J Clin Pharmacol 18: 100- 105, Bei 29 epileptischen Patienten wurde die Konzentra1978. tion en des Phenobarbital (PB) und ihre gegenseitigen Waddell WJ and Butler TC. The distribution and Beziehungen im Gesamtplasma (C,), im plasexcretion of phenobarbital. J Clin Invest maeiweiaungebundenen (Cf)und gemischten Speichel 361217-1226, 1957. (C,) untersucht. Eine hoch signifikante Korrelation wurde zwischen C, und Cr gefunden. Obwohl eine signifikante Beziehung zwischen Cf und C, gefunden R~SUME wurde, betrug das CJCrVerhaItnis nur 0.63. Wenn C, Les relations entre les concentrations de phenobar- korrigiert wurde durch pka (7.41) und das pH des bital (PB) dans le plasma total (C,), dans les proteines Speichels, um die freie PB-Konzentration (C,& f) zu libres du plasma (Cf), et dans les secretions salivaires bestimmen, wurde eine statistisch hochsignifikante mixtes (C,) sont ktudikes chez 29 sujets kpileptiques. Korrelation zwischen Cr und Ces, erreicht; Das Une correlation hautement significative est observee C,,, ,/CrVerhiiltnis war 0.96. Der zeitliche Verlauf von entre C, et C,. Bien qu'une correlation significative soit C,,, aus Cs nach einer einzigen oralen Dosis von SO observkq entre Cf et C,, le rapport C,/Cf n'est que de mg PB bei gesunden mannlichen freiwilligen Erwach0.63. Lorsque C, est comge par l'utilisation de pKa senen wurde als triexponentielle Gleichung geschatzt. (7.41) et du pH de la salive pour apprkcier la concen- Das Maximum wurde 2 Stunden nach Einnahme tration de PB libre (C,,, f), une correlation hautement erreicht und die Halbwertzeit betrug 66 Stunden. significative est obtenue entre Cf et C,,, et le rapport Die Vorhersage der minimalen Konzentration bei C,,, r/Cfest de O.%. L'evolution dans le temps de C,,, einem steady state auf der Basis dieser Parameter obtenu & partir de C,, a p r h une prise orale unique de entspricht sehr wohl den aktuell erzielten C,,, r 50 mg de PB par un volontaire adulte de sexe masculin Werten aufgrund wiederholter Verabfolgung von PB. se prksente approximativement comme une equation (D. Scheffner, Heidelberg) triexponentielle. Le pic maximum se situe 2 heures
Epilepsia, Vol. 20, February 1979
