Introduction

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In general, only the unbound fraction of drugs in media such as blood or protein-containing perfusate is thought to be transferred into body tissues. Thus, unbound fraction values measured with ultrafiltration or equilibrium dialysis in vitro are used not only for measurement of transfer rate into body tissues but also of blood-brain barrier (BBB) permeability. However, when we measured the BBB permeability of diazepam with the intracarotid artery (i.c.a.) injection method, we found that its cerebral concentration was much higher than that estimated based on the assumption that only the unbound fraction measured in vitro is able to penetrate the BBB. Because protein-bound drugs easily dissociate and permeate through the BBB in the cerebral circulation, the concentration of drug in the brain will be higher than that estimated from the unbound fraction in vitro. This suggested that use of the in vitro unbound fraction in determining BBB permeability might not be appropriate. Although this consideration has already been discussed (Pardridge and Landaw, 1984), the BBB permeabilities of most drugs are in fact calculated using in vitro unbound fractions with suitably acceptable results. However, methods based on the theory that drugs binding to protein dissociate rapidly and pass through the BBB may yield more accurate measurements of BBB permeability in vivo.

We therefore used the in situ perfusion method to measure the apparent exchangeable fractions of drugs with BBB permeability too low for measurement with the i.c.a. injection method and compared the results to observe the extent of their agreement.

The drugs used in the present study included diazepam, caffeine, and propranolol, as well as three drugs synthesized in our laboratories: (+)-S-145 Na, a thromboxane A₂ receptor antagonist; S-312-d, a Ca⁺⁺ channel antagonist that improves cerebral blood flow and displays protection of central neurons; and S-8510, a benzodiazepine inverse agonist.

	Experimental Procedures

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Materials. [¹⁴C]Diazepam was obtained from Amersham International (Buckinghamshire, UK). [³H]Diazepam, [¹⁴C]caffeine, L-[³H]propranolol, [³H]glucose, [¹⁴C]sucrose, [³H]sucrose, [³H]water, and [¹⁴C]butanol were obtained from NEN Life Science (Boston, MA). (+)-[¹⁴C]S-145 Na [(+)-[1R-[1a,2a(Z),3b,4a]]7-[3-[[U-¹⁴C]phenylsulfonyl)amino]bicyclo[2.2.1]hept-2-yl]5-heptenoic acid sodium salt], [¹⁴C]S-312-d [(S)-(+)-methyl 3-isobutyl6-methyl-4-(3-nitrophenyl)-4,7-dihydrothieno[2,3-b][4-¹⁴C]pyridine5-carboxylate], and [¹⁴C]S-8510 [2-(isoxazol-3-yl)-3,6,7,9-tetrahydroimidazo[4,5-d]pyrano[4,3-b][4-¹⁴C]pyridine monophosphate monohydrate]were synthesized at Shionogi Research Laboratories (Shionogi & Co., Ltd., Osaka, Japan) and were confirmed to have radiochemical purities above 99% by HPLC. The labeled positions of the test drugs are shown in Fig. 1. All other reagents were of analytical grade.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Compounds synthesized in our laboratories and used in this study include (+)-S-145 Na, a thromboxane A2 receptor antagonist; S-312-d, a Ca++ channel antagonist that improves cerebral blood flow and offers protection of central neurons; and S-8510, a benzodiazepine inverse agonist.

Perfusion Method. Male Sprague-Dawley rats (Clea Japan, Inc., Tokyo) age 9 weeks were used. The rats were anesthetized with pentobarbital; the occipital artery, superior thyroid artery, and pterygopalatine artery were clotted or ligated; and the external carotid artery was cannulated in a retrograde manner with a polyethylene tube (PE50; Intramedic, Sparks, MD) (Takasato et al., 1984). Krebs-Henseleit buffer (118 mM NaCl, 14.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 10 mM D-glucose, pH 7.4) was used as a perfusate. This buffer was bubbled with a mixture of 95% O₂ and 5% CO₂, filtered through Millex HV filter (Millipore, Bedford, MA), incubated at 36°C, and infused with an infusion pump (Harvard Apparatus, S. Natick, MA). For the determination of residual perfusate in the brain, labeled compound and labeled sucrose were added to this perfusate. Immediately after perfusion, the animals were decapitated, and the forebrain was separated from the cerebellum and medulla oblongata. After removal of the pia mater and choroid plexus, the brain was dissolved with tissue solubilizer (Soluen 350; Packard, Meriden, CT), and radioactivity was determined with a liquid scintillation counter (Tri Carb 2200 CA; Packard).

Local Cerebral Perfusate Flow in Rat Forebrain. A mixture of 7.4 kBq (1.03 µg)/ml [¹⁴C]diazepam and 37 kBq (0.017 µg)/ml [³H]sucrose was perfused for 10 s, and the animal was decapitated. Local cerebral perfusate flow (LCPF: represented Q in equation) was obtained using the equation:

(1)

where C_br(T), the parenchymal brain concentration (dpm/g) of [¹⁴C]diazepam at time T was calculated as the measured brain concentration minus intravascular tracer concentration, with the latter equal to the product of regional vascular volume measured by [³H]sucrose multiplied by perfusion fluid concentration of tracer. C_in is the concentration (dpm/g) of unbound [¹⁴C]diazepam in the perfusate, and T is perfusion time (s).

Apparent Exchangeable Fraction in Brain Microcirculation Determined with Perfusion Method. Apparent exchangeable fractions for 3.7 kBq (0.36 µg)/ml [¹⁴C]caffeine, 3.7 kBq (1.3 µg)/ml [¹⁴C]S-312-d, and 37 kBq (18 µg)/ml [¹⁴C](+)-S-145 Na were measured with the perfusion method. Each ¹⁴C-labeled tracer and [³H]sucrose were added to perfusate. The BSA concentration in perfusate was varied between 0 and 1 mM. Perfusion with [¹⁴C]caffeine and [¹⁴C]S-312-d was performed for 10 s, and perfusion with [¹⁴C](+)-S-145 Na was performed for 30 s; the value of C_br(T)/(C_in,tot · T · Q) was obtained for each concentration of BSA. C_br(T) of each tracer was the parenchymal brain concentration. K_d,app was calculated by fitting to eq. 2 (refer to Appendix for details):

(2)

The apparent exchangeable fraction (f_app) was obtained from eq. 14 (Pardridge and Fierer, 1990) given in the Appendix.

Apparent Exchangeable Fraction in Brain Microcirculation Determined with i.c.a. Injection Method. Each rat was anesthetized with pentobarbital, and 0.2 ml of a mixture of test compound and a reference compound was rapidly injected from the carotid artery. The animal was decapitated at 5 s after the injection, and the brain uptake index was calculated from the ratio of test to reference values of the administered solution and that in the brain (Oldendorf, 1970). The test compounds used were 3.7 kBq (0.04 µg)/ml [³H]diazepam, 37 kBq (3.6 µg)/ml [¹⁴C]caffeine, 74 kBq (0.034 µg)/ml [³H]propranolol, 18.5 kBq (6.4 µg)/ml [¹⁴C]S-312-d, 200 kBq(100 µg)/ml [¹⁴C](+)-S-145 Na, and 37 kBq (15 µg)/ml [¹⁴C]S-8510. [³H]Water or [¹⁴C]butanol was used as the reference compound. For comparison of the values obtained with different reference compounds, brain uptake index was converted to the extraction (E) with N-isopropyl-p-iodoamphetamine (Pardridge et al., 1985). The BSA concentration in the administered solution was varied between 0 to 1 mM. E was measured for each concentration, and the apparent dissociation constant (K_d,app ) was calculated by fitting to eq. 3, derived from the model in Fig. 2 (Pardridge and Landaw, 1984):

(3)

where k₃ is the rate constant of drug transport from blood to brain (s¹); t is the mean brain capillary transit time (s); and [P] is the concentration of BSA (M). The apparent exchangeable fraction (f_app) was obtained from eq. 14 (Pardridge and Fierer, 1990) given in the Appendix. The fitting of eqs. 2 and 3 to brain permeability-protein concentration in the perfusate curve was performed with least-squares regression analysis (Yamaoka et al., 1981). The K_{d, app} ± S.D. predicted on the basis of fitting the experimental data to eqs. 2 and 3 is shown in Table 2. The data were analyzed by using the Damping-Gauss-Newton method, except for S-8510 (in the i.c.a. injection method) and caffeine (in the perfusion method), which analyzed with the Simplex method.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   A compartmental model of transport of protein-bound drugs into the brain in vivo during passage through the brain microcirculation. [PD], protein/drug complex; [P], free protein; [D], free drug in the plasma compartment; [Db], free drug in the brain compartment. k1 is the rate constant of drug dissociation (s1), k2 is the rate constant of drug association (mol1 s1), k3 is the rate constant of plasma to brain transport through the BBB (s1), and k4 is the rate constant of brain to plasma transport through the BBB (s1). Because plasma proteins do not cross the BBB, the transport of protein-bound drugs into the brain occurs via a free intermediate mechanism that involves obligatory dissociation of protein-bound drugs into the free intermediate state before BBB transport.

PS Products Determined with Perfusion Method. PS products for 3.7 kBq (0.04 µg)/ml [³H]diazepam, 37 kBq (3.6 µg)/ml [¹⁴C]caffeine, 37 kBq (0.02 µg)/ml [³H]glucose, 3.7 kBq (1.3 µg)/ml [¹⁴C]S-312-d, 37 kBq (18 µg)/ml [¹⁴C](+)-S-145 Na, and 2.5 kBq (1 µg)/ml [¹⁴C]S-8510 were measured using eq. 4 (Takasato et al., 1984). Rats were perfused with buffer containing 0.5 mM BSA at the rate of 3.5 ml/min. Perfusion time for [¹⁴C]S-312-d was 10 s, and that for the other drugs was 30 s. C_br(T) of each tracer was the parenchymal brain concentration. C_in is the concentration of unbound tracer in the BSA-containing perfusate_. In the case of [³H]diazepam, however, the value within parentheses in eq. 4 was negative for half of the measurements, and PS could not be calculated. PS was therefore obtained using eq. 9 in the Appendix from k₃t, which was obtained at the time of the K_{d, app} calculation by using eq. 3.

(4)

Protein Binding In Vitro. Each drug was dissolved in the Krebs-Henseleit buffer containing 0.5 mM BSA (Fraction V; Sigma, St. Louis, MO), and protein binding was measured with the ultrafiltration method using an MPS-3 filter (Amicon, Beverly, MA). In this experiment, the adsorption of each drug to the PMS-3 filter was measured and used to correct the protein binding.

	Results

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LCPF in Rat Forebrain. The flow rate of perfusate from pump was set at three levels (2.5, 3.5, and 5 ml/min), and LCPF was measured in BSA concentrations in perfusate from 0 to 1 mM (Table 1). At each pump flow rate, LCPF in the hemisphere of the forebrain was smaller than the pump flow rate, indicating that perfusate introduced from the carotid artery was sent to several regions other than the forebrain. When the flow rate of perfusate containing 0.5 mM BSA was doubled from 2.5 to 5 ml/min, LCPF increased only about 52%. Thus, deviation of the perfusate outside the forebrain increased when the pump flow rate was elevated. LCPF was little affected by BSA concentrations up to 0.2 mM at any flow rate but gradually decreased at higher concentrations of BSA, probably due to increased vascular resistance resulting from increased viscosity.

Apparent Exchangeable Fraction in Brain Microcirculation Determined with Perfusion Method. Decreases in C_br(T)/(C_in,tot · T · Q) caused by BSA concentration in the perfusate are shown in Fig. 3. The values obtained with the perfusion method are indicated by the symbols, and the values predicted on the basis of fitting the experimental data to eq. 2 are indicated by solid lines. The value of C_br(T)/(C_in,tot · T · Q) for caffeine was not affected by BSA concentrations, that for (+)-S-145 Na sharply decreased, and that for S-312-d showed an intermediate value. The K_d,app of caffeine calculated in this experiment was 19,999 mM, whereas that of (+)-S-145Na was 0.0212 mM (Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Changes in BBB permeability of drugs, Cbr(T)/(Cin,tot · T · Q), with changing BSA concentration in the perfusate in the perfusion method. The BBB permeability of caffeine was not affected by BSA, whereas those of S-312-d and (+)-S-145 Na decreased with increasing BSA concentration. Each symbol represents the mean (n = 5) and S.D.

Apparent Exchangeable Fraction in Brain Microcirculation Determined with i.c.a. Injection Method. The extraction (E) of each drug was determined with the i.c.a. injection method. The experimentally observed values are indicated by the symbols, and the E predicted by fitting the experimental data to eq. 3 is indicated by a solid line (Fig. 4). Although E decreased with increase in BSA concentration in the i.c.a. injection method, the degree of decrease varied depending on the drug. Extraction of S-8510 was not affected by BSA at all, whereas E of diazepam and caffeine were slightly affected and that of (+)-S-145 Na decreased sharply with increasing BSA concentration. The K_d,app of S-8510 was 223,121 mM, whereas that of (+)-S-145 Na was 0.0358 mM, indicating the slow dissociation of latter compound from BSA (Table 2).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Changes in extraction (E) of drugs with changing BSA concentration in the solution administered by the i.c.a. injection method. Although the BBB permeability of S-8510 was low, it was not affected by BSA. The BBB permeabilities of diazepam and caffeine decreased slightly with increasing BSA concentration, whereas those of propranolol, S-312-d, and (+)-S-145 Na in particular markedly decreased with increasing BSA concentration. Each symbol represents the mean (n = 5) and S.D.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Extraction (E) obtained with the i.c.a. injection method and BBB permeability, Cbr(T)/(Cin,tot · T · Q), obtained with the perfusion method at the rate of 3.5 ml/min for (+)-S-145 Na. Although the values of these two indices of BBB permeability differed, the patterns of decrease in BBB permeability with increasing BSA concentration were similar. Each symbol represents the mean (n = 3-5) and S.D.

Comparison of Unbound Fraction In Vitro and Apparent Exchangeable Fraction In Vivo of Diazepam. The unbound fraction of diazepam was extremely decreased by 0.017 mM BSA in the buffer solution in vitro and decreased further as the BSA concentration increased (Table 3). On the other hand, the apparent exchangeable fraction of diazepam was decreased only a little by an increase in the concentration of BSA. In 0.17 mM BSA, the ratio of the apparent exchangeable fraction to the unbound fraction was 3.6, and in 0.5 mM BSA, it was 8; this meant that an 8-fold higher concentration of unbound fraction of diazepam existed in the cerebral circulation and more drug contributed to permeation of BBB under the usual experimental conditions of measuring PS product used in the present study.

PS Products. PS products calculated using the apparent exchangeable fractions measured with the i.c.a. injection method and the perfusion method are compared in Table 4. Diazepam exhibited the highest BBB permeability, 21.3 × 10³ ml/s/g, followed by caffeine, S-312-d, glucose, (+)-S-145Na, and S-8510, in this order. All of the compounds exhibited BBB permeability equal to or higher than that of glucose. Because glucose exhibited no protein binding in the in vitro ultrafiltration method, its unbound fraction was presumed to be 100% in the cerebral circulation in vivo. PS products calculated from the apparent exchangeable fractions obtained with the perfusion method were slightly high for S-312-d and (+)-S-145 Na.

	Discussion

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In general, drugs penetrating into tissues are thought to be those in the unbound fraction in blood, and in vitro values are usually used to estimate drug transport. Detailed analytical methods are available for measuring protein binding through the use of equilibrium dialysis and ultrafiltration. Two protein-binding sites for diazepam and (+)-S-145 Na were indicated with Scatchard plot analysis and Michaelis-Menten plot analysis, and the corresponding dissociation constants, K_d1 and K_d2, of each compound were obtained (Table 2). Caffeine and S-8510 yielded straight lines on Scatchard plots and thus appeared to each have one binding site. In contrast, only the apparent dissociation constant K_d,app can be obtained by fitting data obtained with the i.c.a. injection and perfusion methods. Although the values of K_d1 and K_d,app were almost the same for caffeine and (+)-S-145 Na, they differed greatly for diazepam and S-8510. The significance of these differences is unclear at present. K_d,app was measured for dissociation from albumin in the cerebral circulatory system assuming a single binding site. Although there may be sites with greater or lesser dissociability for compounds from albumin, the K_d,app was measured for overall dissociation from albumin in the cerebral circulation.

Pardridge and coworkers have reported the permeation of protein-bound compounds through the BBB (Cornford et al., 1983; Pardridge et al., 1983; Terasaki et al., 1986). In addition, albumin-bound drugs have been reported to be transported into the liver (Weisiger et al., 1981; Forker and Luxon, 1983).

In the present study, we demonstrated the participation of protein-bound drugs in BBB permeation for diazepam, caffeine, propranolol, S-312-d, (+)-S-145Na, and S-8510 with the i.c.a. injection method and for caffeine, S-312-d, and (+)-S-145 Na with a new analytical procedure of the perfusion method. Although the mechanism by which protein-bound drugs permeate tissues is not known, the report of Horie et al. (1988) that conformational change of albumin occurs in contact with isolated rat hepatocytes is of interest because this would suggest that dissociation of compounds from protein may occur due to the conformational change of albumin when it comes into contact with endothelial cells in brain capillaries. If this does occur, the strength of the hydrogen bonding, which plays an important role in the binding of drugs to albumin, may be related to the apparent exchangeable fraction.

We developed a new method to measure the apparent exchangeable fraction from data obtained with the perfusion method and compared the values with those obtained with the i.c.a. injection method that had already been reported by Pardridge and Landaw (1984). The K_d,app values for (+)-S-145 Na and S-312-d obtained with the perfusion method were nearly the same as those obtained with the i.c.a. injection method. On the other hand, K_d,app for caffeine obtained with the perfusion method was very large and differed greatly from that obtained with the i.c.a. injection method (Table 2). However, the apparent exchangeable fractions (f_app) of caffeine calculated from K_d,app were 100% for the perfusion method and 90.9% for the i.c.a. injection method and thus differed only a little. In the case of caffeine, which undergoes less dissociation from BSA and has a large apparent exchangeable fraction, the different K_d,app may have been predictable on the basis of fitting estimation from different experimental methods.

The K_d,app obtained under anesthesia with ether was smaller than that obtained with pentobarbital because blood flow is more rapid under ether anesthesia (Pardridge and Fierer, 1990). If this is true, it should not be possible to apply the apparent exchange fraction obtained with the i.c.a. injection method to the perfusion method when LCPF is larger than the cerebral blood flow of the i.c.a. injection method. Therefore, the K_d,app for (+)-S-145 Na was measured by changing the perfusion rate (Fig. 6). The effects of BSA on the C_br(T)/(C_in,tot · T · Q) for (+)-S-145 Na were unchanged when the perfusion velocity was increased from 2.5 to 5 ml/min, and no difference in K_d,app was found. These observations confirmed that K_d,app was not affected by a change in the blood flow rate.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of changes in perfusion rate on the BBB permeability, Cbr(T)/(Cin,tot · T · Q), induced by changing the concentration of BSA in the perfusate. When the perfusion rate was increased to 2.5, 3.5, or 5 ml/min, the decrease in BBB permeability caused by BSA remained the same, indicating that the perfusion rate had no effect on the apparent exchangeable fraction. Each symbol represents the mean (n = 3-5) and S.D.

In conclusion, the following findings validated the use of the perfusion method for determination of the apparent exchangeable fraction: 1) the apparent exchangeable fractions obtained with the i.c.a. injection method reported by Pardridge and colleagues were equivalent to those obtained with our perfusion method, 2) the effect of BSA on C_br(T)/(C_in,tot · T · Q) measured with the perfusion method was similar to that on E measured with the i.c.a. injection method, and 3) equal and stable values of K_d,app were obtained with different perfusion velocities.