Critical factors of intracerebral microdialysis as a technique to determined the pharmacokinetics of drugs in rat brain

BRAIN RESEARCH ELSEVIER

Brain Research 666 (1994) 1-8

Research report

Critical factors of intracerebral microdialysis as a technique to determined the pharmacokinetics of drugs in rat brain Elizabeth C.M. de Lange *, Meindert Danhof, Albertus G. de Boer, Douwe D. Breimer Leiden/Amsterdam Centerfor Drug Research, Division of Pharmacology, Sylvius Laboratory, P.O. Box 9503, 2300 RA Leiden, The Netherlands

Accepted 13 September 1994

Abstract

The purpose of this investigation was to determine the effect of experimental conditions on the concentrations of atenolol and acetaminophen in brain microdialysate, and to investigate the feasibility of performing repeated experiments within individual rats. Following intravenous bolus administration, reproducible concentration-time profiles were obtained in plasma and in brain dialysate. Based on corrections for in vitro recoveries of the intracerebral probe, the estimated ratio of the AUC in brain extracellular fluid (AUCbrai n ECF) over the AUC in plasma (AUCplasrna) +S.E.M. was 3.8 +0.6% (n =6) for atenolol and 18 + 2% (n = 6) for acetaminophen. Upon intracerebroventricular administration, interanimal differences in kinetics of acetaminophen in brain dialysate were observed while the concentrations of atenolol were below the detection limit of the assay. The influence of the use of isotonic versus hypotonic perfusate solutions o n mUCbrain ECF values after intravenous bolus administration of both drugs was determined. Repeated experiments with the isotonic perfusate (24, 48 and 78 h post-surgery) resulted in mUCbrai n ECF values with the ratio of 100: 98: 76% for acetaminophen and 100: 103: 98% for atenolol. Using a hypotonic perfusion solution the ratio of AUCbrai n ECF values was 100: 154: 114% for acetaminophen and 100: 378: 427% for atenolol. A clear effect of the temperature of the hypotonic perfusate (24 vs 38°C) on acetaminophen AUCbrai . ECF values was revealed. The ratio of mUCbrai n EcFValues obtained at 24: 38°C was 192: 100%. It was concluded that intracercbral microdialysis can be used to study the pharmacokinetics of drugs in the brain, provided that experiments are performed under carefully controlled conditions. Keywords: Intracerebral microdialysis; Condition; Brain; Rat; Acetaminophen; Atenolol; Pharmacokinetics; Blood-brain barrier

I. Introduction

The b l o o d - b r a i n barrier (BBB) plays a major role in the pharmacokinetics of drugs with an action on the brain. Especially for relatively hydrophilic drugs, the transport from blood to the brain is restricted [7,28,29]. Several factors (including diseases) have been shown to alter the BBB permeability [2,27], and have to be taken into consideration in pharmacodynamic studies with drugs acting on the brain. Several in vivo experimental methods have been used to study BBB transport [9,16]. In general they can be divided into single and multiple passage techniques, whereby mostly a comparison is made between blood

* Corresponding author. Fax: (31) (71) 276292. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01130-3

and tissue drug concentrations at a single point in time, following systemic administration. A limitation of these methods is that only a single concentration point is obtained within each individual animal. This problem has been overcome in the technique used by van Bree et al. [28]; by means of serial cerebrospinal fluid (CSF) and blood sampling, the time concentration profile of a drug at both sampling sites can be determined within one animal, and compared. However, CSF concentrations of a drug do not per se reflect the concentrations in different parts of the brain parenchyma [13,18]. Potentially, local concentration measurements can be performed using intracerebral microdialysis. This technique involves the stereotactic insertion of a microdialysis probe into a selected brain area. A physiological solution is p u m p e d through the probe. Small molecular weight molecules dialyse through the membrane and are carried out by the moving fluid which

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E.C.M. de Lange et al./Brain Research 666 (1094) 1--8

s u b s e q u e n t l y can be collected a n d analyzed. T h e conc e n t r a t i o n of a c o m p o u n d in the dialysate supposedly reflects the c o n c e n t r a t i o n in the extracellular fluid s u r r o u n d i n g the m e m b r a n e of the microdialysis p r o b e [5]. Initially, this t e c h n i q u e was mostly applied to m o n i tor c o n c e n t r a t i o n s of e n d o g e n o u s c o m p o u n d s in n e u r o chemical studies. A l t h o u g h elegant a n d simple in principle, microdialysis is an invasive t e c h n i q u e . Localized tissue reactions to the i m p l a n t a t i o n of foreign bodies have b e e n described [22]. They include n e u r o n a l damage, microglial a n d astroglial reactions, localized h e m a t o m a s , alterations in glucose metabolism, alterations in BBB functionality a n d local biochemical disturbances. T o some extent the reactions on the i m p l a n t a t i o n of the microdialysis probe have b e e n investigated. Initially, formation of eicanosoids [31], local d i s t u r b a n c e s in cerebral blood flow and glucose m e t a b o l i s m were f o u n d [4], which were more or less n o r m a l i z e d one day after surgery. Studies on BBB integrity showed that despite some degree of b r a i n tissue t r a u m a , the BBB a r o u n d the probe was intact shortly after i n s e r t i o n [4]. Histological e v a l u a t i o n revealed that glial reactions usually started two or three days after i m p l a n t a t i o n [3]. Furt h e r m o r e , it was f o u n d that quite different results can be o b t a i n e d on the (pharmacologically stimulated) release of n e u r o t r a n s m i t t e r s , d e p e n d i n g o n the type a n d size of the p r o b e [20,30], the post-surgery interval [20], and the Ca 2÷ c o n t e n t of the perfusate [14]. It is clear that e x p e r i m e n t a l c o n d i t i o n s have to be c o n s i d e r e d as well in p h a r m a c o k i n e t i c studies to which the use of i n t r a c e r e b r a l microdialysis has b e e n ext e n d e d more recently [15,23,24]. For studies dealing with the t r a n s p o r t of drugs across the BBB, insight has to be gained first a b o u t the possible effects on BBB p e r m e a b i l i t y (and therefore on the dialysis outcomes) by i m p l a n t a t i o n of the p r o b e a n d the e x p e r i m e n t a l conditions used. In the p r e s e n t study we have examined the effect of some e x p e r i m e n t a l c o n d i t i o n s of the i n t r a c e r e b r a l microdialysis t e c h n i q u e on the p h a r m a cokinetics of drugs in cortical b r a i n dialysate of individual rats. Specific a t t e n t i o n has b e e n paid to factors that have not b e e n investigated in the a b o v e m e n t i o n e d studies, like perfusate t e m p e r a t u r e a n d differences between tonicity of the perfusate a n d b r a i n ECF. Ace t a m i n o p h e n a n d atenolol were used as model drugs, r e p r e s e n t i n g a m o d e r a t e l y lipophilic a n d a hydrophilic drug, in order to see w h e t h e r n o r m a l BBB t r a n s p o r t characteristics would be o b t a i n e d w h e n using this technique. C o n c e n t r a t i o n - t i m e profiles of these drugs in dialysate a n d plasma were o b t a i n e d after i n t r a v e n o u s (i.v.) a n d i n t r a c e r e b r o v e n t r i c u l a r (i.c.v.) a d m i n i s t r a t i o n . T h e i n f l u e n c e of the osmolality a n d t e m p e r a t u r e of the perfusate on the extent of drug t r a n s p o r t into the b r a i n was studied, as well as the possibility to p e r f o r m rep e a t e d e x p e r i m e n t s in individual rats.

2. Materials and m e t h o d s 2.1. ,4nimals

Adult male SPF Wistar rats (body weight 180-200 g) of the Sylvius laboratories, University of Leiden, were used. The rats were housed in Macrolon cages on standard hard wood bedding with free access to water and standard laboratory rat diet (RMH-TH, Hope Farms, Woerden) 2.2. Surgery

For microdialysisthe rats were anaesthetized with an intramuscular injection of 150 /xl of HYPNORM R (Janssen Pharmaceutica, Goirle, The Netherlands) and placed in a stereotaxic frame. Incisions were made to expose the skull which was thereafter locally anaesthetized with a 0.6% solution of lidocaine. 1.5 mm holes were drilled in the lateral plane of the skull, allowing the horizontal introduction of a dialysis probe, using a tungsten wire (TW5-3, Clark Electro Medical Instr., UK), through the cortex at 2 mm directly below bregma. The dialysis fibre (o.d. 0.29 mm, C-DAK artificial kidney 201-800 D 135 SCE, CD Medical B.V., Rotterdam, The Netherlands) was covered with silicon glue (Rhodosil CAF 3, Rhone-Poulenc, Amstelveen, The Netherlands) except for a 10 mm central zone. Stainless-steel needles, glued to both ends of the dialysis fibre, were secured with dental cement on the top of the skull. The animals were allowed to recover from probe implantation and anaesthesia for 24 h before the start of the first experiment. A bulk perfusate temperature of 38°C was achieved by the use of a subcutaneous cannula (polyethylene tube, i.d. 0.58 mm, lenght of about 20 cm) at the back of the rat. The perfusate fluid was led through this cannula, allowing the fluid to equilibrate to rat body temperature, before entering the microdialysisprobe in the brain. For i.v. drug administration and serial blood sampling polyethylene cannulas (i.d. 0.58 mm, o.d. 0.8 mm) were implanted into the femoral vein and femoral artery respectively under ether anaesthesia. The recovery period was at least 2 h before the start of the experiment. For the i.c.v, administration of a drug, a cannula was placed in the left lateral ventricle immediately before the insertion of the microdialysis probe: a hole was drilled using a 1 mm dental burr, at 1.5 mm lateral and 0.8 mm rostral from bregma. A polyethylene tube (I.D 0.4 mm and O.D 0.8 mm) with a depth marker at 3.2 mm from the cannula tip, was inserted into the lateral ventricle and immediately fixed with a drop of cyanoacrylate glue. 2.3. Experimental procedure

For transcortical microdialysis experiments, the stainless steel needles at both sides of the microdialysis probe were connected by means of polyethylene tubing (o.d. 0.51 mm, i.d. 0.08 mm, 80 cm) to a perfusion pump (Gilson) and a sample loop respectively.The latter was connected to an HPLC. The dialysis probe was perfused at 7 /xl/min with a medium consisting of 145 mM NaC1, 0.6 mM KC1, 1.0 mM MgCI2, 1.2 mM CaCI2, 0.2 mM ascorbic acid, in a 2 mM potassium phosphate buffer, pH = 7.4 [14]. The composition of the hypotonic perfusate was identical to the isotonic perfusate, except for the NaCI content, which was 14.5 mM, leaving this solution a factor 9 lower in tonicity. The rats were put into the experimental cage, and were dialyzed with buffer solution for 30 min to obtain dialysis equilibrium and blank data. Subsequently the drug was administered and dialysate samples were measured for 120 min. During the course of the experiment the rats were freely moving and had free access to water and food.

E.C.M. de Lange et al. / Brain Research 666 (1994) 1-8 For i.v. administration the drug solution was injected into the femoral vein over 1 min. A solution of 825 /~g (5.4 /~mol) of acetaminophen (neutral) or 10 mg (38 tzmol) of atenoiol (base) in 500 /zl saline (neutralized with HCI) was administered. At regular intervals (0, 5, 15, 30, 45, 60, 90, and 120 min) blood samples of 200 /zl were drawn from the femoral artery and heparinized. Plasma was obtained by centrifugation and stored at -20°C until analysis. In the consecutive experiments individual rats were used repeatedly every day, with the experiments started at post surgery intervals of 24, 48 and 72 h. The i.c.v, administration of drug occurred via the ventricular cannula over 5 s. A solution of 210/zg (1.4/zmol) of acetaminophen or 150 ~zg (0.6 ~zmol) of atenolol in respectively 10 or 15/zl of saline was administered. The total volume of the drug solution administered was corrected for the dead volume of the cannula. At the end of the experiment the position of the cannula in the lateral ventricle was verified by injection of 5 ~1 of 0.5% Evans Blue solution in saline. The rat was then decapitated, the skull opened, the brain removed and dissected by a paramedial coronal section. Blue coloration of the entire ventricle system indicated a correct cannula position.

2.4. Drug analysis 2.4.1. Acetaminophen The acetaminophen HPLC system consisted of a reversed phase column (Spherisorb, 10 cmx4.6 mm i.d., $ 3 0 D S 2, Phase Separations, Waddinxveen, The Netherlands), a precolumn (PeUicular reverse phase, Chrompack) and a electrochemical detector (glassy carbon electrode with an oxidation potential of 800 mV versus an Ag/AgCI electrode, Antec Leyden B.V., The Netherlands). The mobile phase consisted of a mixture of a 20 mM sodium phosphate and a 50 mM sodium citrate buffer containing 0.25 mM of sodium octane sulfonic acid and 15% (v/v) of methanol, pH = 3.0. The flow was 1.0 ml/min. Chromatographic data were recorded and processed with a SP4100 computing integrator (Spectra Physics, SP4100 computing injector, Spectra Physics B.V., Eindhoven, The Netherlands). The interassay variability in aqueous solution was less than 3% (n = 15), with a detection limit of 0.1 ng/ml (66 fmol). All assay calibration curves showed good linearity (correlation coefficient > 0.999). The microdialysate was on-line injected, up to 120 min after administration of the drug, into the HPLC system through a Valco injection valve equipped with a 10/zl loop, with a repetition time of 3.5-4.0 rain. For the analysis of the plasma samples 100 jzl of 6% (w/v) perchloric acid was added to 50/zl of plasma and vortexed for 20 sec. An aliquot of 90/~1 of the supernatant obtained from centrifugation was mixed with 70 p.I of 1 N sodium acetate. With the aid of an autosampler (SP8780 XR, Spectra Physics) 50 p.1 was injected in the HPLC system.

2.4.2. Atenolol The atenolol HPLC system consisted of a reversed phase column (Spherisorb, 10 cm×4.6 mm i.d., $ 3 0 D S 2, Phase Separations, Waddinxveen, The Netherlands), a precolumn (Pellicular reverse phase, Chrompack, Middelburg, The Netherlands) and a Shimadzu RF-350 Fluorimeter (Shimadzu Corp., Kyoto, Japan) with an excitation wavelength of 276 and an emission wavelength of 309 nm. The mobile phase consisted of 73.5% (v/v) sodium acetate buffer pH = 4.0 containing 5 mM of sodium octane sulfonate and 26.5% (v/v) of acetonitrile. The flow was 1.0 ml/min. The inter-assay variation in aqueous solution was less than 7% (n = 15), with a detection limit of 100 ng/ml (6 pmol). All assay calibration curves showed good linearity (correlation coefficient > 0.999).

3

The microdialysate was on-line injected, up to 120 min after administration of the drug, into the HPLC system by a Valco injection valve equipped with a 16/zl loop, with a repetition time of 3.5-4.0 min. To 100 /zl of plasma 100 /zl of a pindoiol solution (25 /~g/ml water) were added as internal standard. Furthermore, 100/zl of 4 M sodium hydroxide and 0.9 ml of water were added to the sample. Subsequently the aqueous mixture was extracted with 5 ml of ethyl acetate. After centrifugation, the aqueous layer was discarded and 200 ~1 of 1% (w/v) phosphoric acid was added to the organic layer; tubes were shaken vigorously for 30 s. The layers were separated by centrifugation and the organic layer was discarded. To remove ethyl acetate traces from the aqeous layer, the sample was put in a vacuum evaporator (Vacuum vortex, Buchler, Ford Lee, NY, USA) for 15 min. From the remaining residue, 50/zl was injected into the HPLC system.

2.5. Dam ana~s~ The profiles obtained from the dialysate were corrected for the time needed to transport the dialysate into the HPLC system (lagtime). In vitro recovery values were used to estimate Cbrai n ECF from the Cdial values of the drugs, because an appropriate method to determine in vivo recovery under non-steady-state conditions experimentally was not available. In vitro recovery at 37°C was detrmined for a 10 mm dialysis zone in a non-stirred solution of the drug in the perfusate buffer. This was (mean+S.E.M.) 23_+ 1% for acetaminophen and 13+ 1% for atenolol (n = 3 probes, 3 determinations per probe). Individual Cbrai n ECF and Cplasma of acetaminophen and atenolol were used to calculate AUC values (0-120 min) by means of the trapezoidal rule (Siphar, modeling package, SIMED, Creteil, France). The terminal elimination rate constants were determined from the terminal linear part of the curves. Tabel 1 gives the mean-+ S.E.M. values of individually determined pharmacokinetic parameters. The AUC values were used to compare the results. Statistical evaluation was performed with Student's t-test (to compare mean data) or Duncans Multiple rank test (for paired data in the repeated experiments) for P < 0.05.

3. Results

3.1. Pharmacokinetics of acetaminophen and atenolol in plasma and brain dialysate Following i.v. administration, the Cplasma and Cbrai n ECF of acetaminophen were obtained, as shown in Fig. 1A. Between animals little variation was observed between the individual concentration-time profiles. The resulting pharmacokinetic parameters are presented in Table 1. The terminal elimination half-live ( + S.E.M.) of acetaminophen in the brain extracellular fluid was not significantly different from that in plasma, and was 16.5 + 3.5 min and 22.4 :t: 4.3 respectively. Comparing AUC values, it was found that A U C b r a i n ECF was 18 _+ 2% of t h e AUCplasma. I.c.v. administration of acetaminophen resulted in quite reproducible Cplasmawhile the Cbrai. ECF revealed a wide inter-animal variability (Fig. 1B,C). In Fig. 2, the Cplasma and Cbrain ECF of atenolol following i.v. administration are given. The pharma-

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E.C.M. de Lange et al./Brain Research 666 (1094) 1-8

0.1

cokinetic parameter estimates of atenolol are given in Table 1. Only the AUCbrai n ECF was significantly different from A U C p l a s m a . The amount of atenolol reaching the brain, as reflected by the ratio of the A U C b r a i n l~(:b over AUCplasma ( ± S.E.M.) was 3.8 + 0.6%. Following i.c.v, administration, no concentrations of atenolol in either plasma or brain dialysate could be detected with the current atenolol assay.

0.01

3.2. Effect of isotonicity and hypotonicity of the perfuste on AUCbr~i,, ECF in repeated experiments

10 I A

1

c O rO_ 0 C ~D

0.001 0

30

60

90

120

tlrne (rain)

10

1

0.1 0 c

In order to determine whether reproducible Cbrai n ECF could be obtained in daily performed experiments, profiles of acetaminophen and atenolol in brain ECF were measured following i.v. administration on three consecutive days (at PSI of 24, 48 and 72 h) within the same rat, using the isotonic perfusate. The relative values of A U C b r a i n ECF, with reference to the value on the first day, are given in Fig. 3. For acetaminophen, in the third experiment a significantly lower A U C b r a i n ECF was observed. For atenolol, no significant difference in the AUCbrain ECF was observed on these occasions. When using a hypotonic perfusion solution and i.v. administration of acetaminophen, more variable values were observed on the three consecutive days. With atenolol, a profound increase in A U C b r a i n ECF was observed on the second and third day (Fig. 3).

E-

co L~

3.3. Effect of temperature and iso- or hypotonicity of the perfusate on AUCbrai n ECF

0.01

0.001 0

30

60

90

120

time (mln)

10

For both atenolol and acetaminophen the effect of perfusate temperature (24 and 38°C) o n Cbrain ECF was determined. The AUfbrai n ECF values in these experiments are given in Fig. 4. For acetaminophen and atenolol no difference between the AUCbrain ECF values was observed for the isotonic perfusate. For acetaminophen, a larger m U C b r a i n ECF was found at a perfusate temperature of 24°C under hypotonic conditions (Fig. 4).

1

5. Discussion

01

cc-

The purpose of this investigation was to examine the effects of experimental conditions of intracerebral microdialysis on the pharmacokinetic profiles of two model drugs in cortical brain dialysate. Acetaminophen

0.1

O c-

O.01

0.001 0

30

60 tJrne (mJn)

90

120

Fig. 1. Individual (A) Cplasrna ( * , n = 6) and C b r a i n ECF (0, n = 6) of acetaminophen after i.v. administration of 825 ~g; (B) C plasma(g/ = 6); and (C) C b r a i n ECF ( n = 6 ) Of acetaminophen after i.c.v, administration of 210 izg (isotonic perfusate at 38°C, post surgery interval of 24 h).

E.C.M. de Lange et al. / Brain Research 666 (1994) 1-8

5

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0.01 0

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90

120

i: 100

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Fig. 2. Individual Cplasma ( * , n = 5) and Cbrai n ECF (e, n = 6) of atenolol after i.v. administration of 10 rag; after i.c.v, administration of 150/zg, atenolol concentrations in the plasma and dialysate were below the detection limit of the assay (isotonic perfusate at 38°C, post surgery interval of 24 h).

and atenolol were chosen as model drugs because of their different lipophilicity (Table 2) and BBB transport [28]. The results suggest that in principle the microdialysis technique opens interesting perspectives to determine BBB transport characteristics of drugs, provided that experimental conditions are well-controlled. Intracerebral microdialysis has already been applied frequently to monitor concentrations of endogenous compounds in neurochemical studies. It has recently

24 post

48 surgery

72

~nterval (hourS)

Fig. 3. Influence of isotonic (iso, n = 6) versus hypotonic (hypo, n = 6 ) perfusate (38°C) on mWCbrain ECF values of both acetaminophen (AC) and atenolol (AT) after i.v. administration, in repeated experiments after a post surgery interval of 24, 48 and 72 h; the value of the mUCiso, 24 h has been taken as 100%. Values are presented as m e a n ± S.E.M. * Significantly different (Student's t-test, P < 0.05) compared to the accompanying mUfbrai n ECF values obtained at a post surgery interval of 24 h.

been extended to the field of pharmacokinetics [15,23]. These studies have mostly been performed in anaesthetized animals, more or less directly after the implan-

Table 1 Pharmacokinetic parameters of acetaminophen and atenolol after i.v. and i.c.v, administration (isotonic perfusate at 38°C, post surgery interval 24 h) Plasma

Brain E C F

Plasma

Brain E C F

i.v. 825 75.2 + 2.4 0.028 + 0.005

i.v. 825 13.8 _+ 0.5 * 0.034 + 0.009

i.c.v, 210 17.8 + 0.6 * * 0.029 ± 0.004

i.c.v. 210 15.6 + 7.4 0.029 + 0.007

390 + 57

NA

503 + 111

NA

11 + 0.4

NA

13 _+ 1.4

NA

Acetaminophen Administration Dose (/zg) A U C (/zg- m i n / m l ) Terminal elimination rate constant (min - 1) Total volume of distribution (ml) Total clearance ( m l / m i n )

Atenolol Administration Dose (mg) A U C (/~g. m i n / m l ) Terminal elimination rate constant (min - t) Total volume of distribution (ml) Total clearance ( m l / m i n )

i.v. 10 1635 + 149 0.019 + 0.003 326 + 28.5 6.2 + 0.44

i.v. 10 62.4 ± 12.8 * 0.015 ± 0.001 NA NA

Values are presented as m e a n + S.E.M. (n = 6). * Significantly different (Student's t-test, P < 0.05) vs. corresponding plasma values. * * Significantly different (Student's t-test, P < 0.05) vs. plasma value after i.v. administration. NA, not applicable.

(~

E.C.M de Lange et al. /Brain Research 066 (1994) 1-8

25O

[

i

200

150

AC, 1SO AT, 1SO [~----J

AC, hypo

100 +

50

-

I 0

24 laerfusate

38 temperature ('C)

Fig. 4. Influence of the perfusate temperature (24 versus 38°C) on AUCbrain ECF values of acetaminophen (AC) and atenolol (AT) after i.v. administration, as obtained with the isotonic (iso, n = 6) and hypotonic (hypo, n = 6) perfusate (post surgery interval of 24 h). Values are presented as mean + S.E.M. * Significantly different (Student's t-test, P < 0.05) compared to the AUCbrai n ECF values obtained at 38°C.

tation of the dialysis probe into the brain tissue. Our experiments with acetaminophen and atenolol were performed in freely moving rats, 24 h after implantation of the transcortical probe. This was assumed to be the optimal time to perform the experiments. It seems that at that time the tissue has recovered from initial reactions on the implantation of the probe, as measured by the formation of eicosanoids [31] and changes in cerebral blood flow [4]. On the other hand, the long-term reactions that have been reported [4,30], are considered not to be of importance at 24 h post surgery. The results obtained at a PSI of 24 h were, therefore, used as reference values in the investigation on the effects of different experimental conditions. Table 2 Properties of acetaminophen and atenolol Drug

Atenolol

Acetaminophen

Molecular weight log P (octanol) a pKa % plasma protein binding % ionized at pH = 7.4

266 - 1.78 9.5 b +_5 b 99

152 0.25 9.6 c + 25 d, 0-15 e 99

a [281; b [251; c [191; a [1]]; e [12].

Following i.v. bolus administration, the plasma pharmacokinetic parameters of acetaminophen [15,17,28] and atenolol [26,28] arc within the same order of magnitude as those previously reported. For both drugs, the resulting Cbram ECF were well-defined (Figs. IA and 2), and considerably lower than AUCp] . . . . values. It has been reported that with the microdialysis technique in principle free (i.e. unbound) concentrations are measured [5]. In the present investigation, total concentrations in plasma have been determined versus free concentrations in brain extracellular fluid. It is unlikely that the difference in concentrations between plasma and brain ECF can be explained by binding to plasma proteins, since plasma protein binding of both drugs has been reported to be low (Table 2). Lower brain ECF concentrations are, therefore, supposed to reflect the restricted BBB transport, which becomes increasingly important with increase in lipophilicity of the drug [16]. Besides lipophilicity, molecular size [29] and charge are also factors which determine diffusional transport across the BBB. Therefore, acetaminophen is expected to diffuse across the BBB to a larger extent than atenolol, which is consistent with the obtained results. The brain microdialysis concentrations as obtained here can also be compared more directly to the results of other studies. For acetaminophen the brain microdialysis profiles as obtained by Morrison et al. [15] showed similar kinetics. With respect to atenotol it was reported that this drug was widely distributed to most body tissues while only a small fraction of the single dose reached the brain [1,10,25]. The ratios between plasma and brain that can be derived from these studies were about 5%, and our value (3.8%) is in line with these findings. A point to consider, however, is that the recovery which is used to estimate brain ECF concentrations from the dialysate data, has been determined in an in vitro experiment and may not accurately predict the recovery in vivo [5,8,15,23]. However, in a following study (unpublished results), it was found that for both drugs the in vivo recovery was at maximum two-fold lower than the in vitro recovery. So, the conclusion may be drawn that the BBB transport for acetaminophen is larger than that for atenolol. Following i.c.v, administration, in the present microdialysis studies no atenolol concentrations could be measured in brain ECF, while acetaminophen concentrations varied widely (Fig. lb). A comparison could be made with results on CSF sampling obtained in this laboratory, using the same breed of rats, dosage, anaesthesia and post surgery interval [28]. The concentration-time profiles of both acetaminophen and atenolol in CSF following i.c.v, administration were rather reproducible. This indicated a difference between brain ECF and CSF concentrations following i.c.v, adminis-

E.C.M. de Lange et al. / Brain Research 666 (1994) 1-8

tration. It was hypothetized that intracerebral microdialysis measurements were very local, and therefore reflected (microscopic) interanimal variation in distance between microdialysis probe and ventricle. This hypothesis was based on previous reports on spatial concentration gradients over brain tissue upon intraventricular administration [6] or microperfusion [21], and has been proven valid in a following study (unpublished results). In intracerebral microdialysis experiments possible reactions on the implantation of the probe, like gliosis, are time-dependent [3]. In addition these reactions may be influenced by the dialysis procedure itself, and the choice of the perfusate may be an important factor. Deviations in its composition as compared to brain ECF may have important effects on functionality of the tissue under examination. With regard to the calcium concentrations, clear alterations of the basal outflow and pharmacological responsiveness of striatal dopamine have been observed [14]. In this study the feasibility of longitudinal experiments was investigated at three consecutive days within the same rat, following i.v. administration of both drugs, using a hypotonic or a isotonic perfusate. The hypotonic perfusate, with important differences in sodium and chloride ion concentrations as compared to the brain ECF, was used to disturb the tissue under investigation locally, and was expected to affect the microdialysis results. These results could then be compared to the data obtained with the isotonic medium which was supposed to be physiological. It was found that the repetitive use of the hypotonic perfusate (38°C) indeed altered the data obtained; a marked increase in m U C b r a i n ECF values was found (Fig. 3). The increase in m U C b r a i n ECF was most pronounced for the hydrophilic drug atenolol. This may be indicative of increased BBB permeability, because many diseases (in this study the disturbance of ion concentrations) can affect BBB functionality, and substances that are normally more or less excluded may enter the brain to a larger extent. When using the isotonic medium (38°C), rather reproducible AUC values were found, which indicates that the microdialysis experiments performed under this condition may be considered as being physiological and therefore reflect 'true' brain ECF concentrations. Until now, most microdialysis experiments are performed with the bulk perfusate at room temperature. Therefore, a temperature gradient exists when the perfusate enters the microdialysis probe. It is, therefore, very well possible that in the periprobe tissue a temperature gradient will exist at the level of dialyzing part of the probe, because the fluid may not be warmed effectively to body temperature at that time. The effect of perfusate temperature, before entering the probe (24 ° versus 38°C), on pharmacokinetics of acetaminophen and atenolol in brain ECF was assessed.

7

No temperature effect could be demonstrated under normal conditions (Fig. 4). However, with the use of a hypotonic dialysate, a clear temperature effect was observed for acetaminophen (Fig. 4). It is possible that the periprobe tissue, already 'stressed' by the hypotonic condition, loses its capability to abolish temperature effects, resulting in a change with respect to permeability a n d / o r diffusion characteristics of the tissue. This observation may have important implications in case pathological ('stressed') tissue will be investigated. Although the observed effects on hypotonicity and temperature of the dialysate will probably be more pronounced within the present experimental design (transversal linear probe, flow rate 7/zl/min) as compared to the more common used (1-shaped probe, flow rate 2 ~l/min), it is the authors' opinion that the use of the (obviously isotonic) perfusate at 38°C is preferable for every microdialysis study. In conclusion, these experiments show the potential use of intracerebral microdialysis in obtaining pharmacokinetic parameters of drugs in brain ECF. Drug dependent changes in the extent of drug transport into the cortex could be detected under various experimental conditions. In addition, indications for a local concentration difference between CSF and brain ECF for acetaminophen and atenolol were found. Repeated experiments could be performed (for each drug to be determined), which opens up the possibility for longitudinal studies. Furthermore, a clear perfusate temperature effect in an induced pathophysiological state was demonstrated. This stresses the importance of carefully controlled conditions when using intracerebral microdialysis. Experiments performed with a perfusate temperature of 38°C are expected to result in the best possible reflection of the physiological situation.

Acknowledgements The authors are greatful for the technical assistence of Mariska Langemeijer and Erica Tukker. This work was supported in part by the Dutch Cancer Foundation.

Abbreviations AUC AUC brainECF A U C plasma

BBB C C brain ECF

area under concentration-time curve AUC in brain dialysate after correction for in vitro recovery AUC in plasma blood-brain barrier concentration-time curve concentration-time profile in brain dialysate after correction for in vitro recovery

E.C.M. de Lange et al. / Brain Research 666 (1994) 1-~' Cdial C pl~lsma

CNS CSF ECF i.c.v. i.d. i.v. log P o.d.

PSI w/v

concentration-time curve in dialysate concentration-time profile in plasma central nervous system cerebrospinal fluid extracellular fluid intracerebroventricular internal diameter intravenous log partition coefficient octanol:aqueous buffer pH = 7.4 outer diameter post surgery interval weight/volume

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