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. 1999 May;43(5):1091–1097. doi: 10.1128/aac.43.5.1091

In Vivo and In Vitro Toxicodynamic Analyses of New Quinolone-and Nonsteroidal Anti-Inflammatory Drug-Induced Effects on the Central Nervous System

Hideki Kita 1, Hirotami Matsuo 1, Hitomi Takanaga 1, Junichi Kawakami 2, Koujirou Yamamoto 2, Tatsuji Iga 2, Mikihiko Naito 3, Takashi Tsuruo 3, Atsushi Asanuma 4, Keiji Yanagisawa 4, Yasufumi Sawada 1,*
PMCID: PMC89116  PMID: 10223919

Abstract

We investigated the correlation between an in vivo isobologram based on the concentrations of new quinolones (NQs) in brain tissue and the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) for the occurrence of convulsions in mice and an in vitro isobologram based on the concentrations of both drugs for changes in the γ-aminobutyric acid (GABA)-induced current response in Xenopus oocytes injected with mRNA from mouse brains in the presence of NQs and/or NSAIDs. After the administration of enoxacin (ENX) in the presence or absence of felbinac (FLB), ketoprofen (KTP), or flurbiprofen (FRP), a synergistic effect was observed in the isobologram based on the threshold concentration in brain tissue between mice with convulsions and those without convulsions. The three NSAIDs did not affect the pharmacokinetic behavior of ENX in the brain. However, the ENX-induced inhibition of the GABA response in the GABAA receptor expressed in Xenopus oocytes was enhanced in the presence of the three NSAIDs. The inhibition ratio profiles of the GABA responses for both drugs were analyzed with a newly developed toxicodynamic model. The inhibitory profiles for ENX in the presence of NSAIDs followed the order KTP (1.2 μM) > FRP (0.3 μM) > FLB (0.2 μM). These were 50- to 280-fold smaller than those observed in the absence of NSAIDs. The inhibition ratio (0.01 to 0.02) of the GABAA receptor in the presence of both drugs was well-fitted to the isobologram based on threshold concentrations of both drugs in brain tissue between mice with convulsions and those without convulsions, despite the presence of NSAIDs. In mice with convulsions, the inhibitory profiles of the threshold concentrations of both drugs in brain tissue of mice with convulsions and those without convulsions can be predicted quantitatively by using in vitro GABA response data and toxicodynamic model.

Many clinical cases, clinical tests, and studies involving tests with animals of the oxidative interaction between new quinolones (NQs) and other drugs have been reported. The toxicity induced by the inhibition of metabolism by caffeine (3, 21, 22) or theophylline (14, 19, 30, 31) and the remarkable reduction in the intestinal absorption of NQs by the interaction between NQs and the metal cations (Ag3+, Mg2+, etc.) in antacids or anti-peptic ulcer drugs (5, 18) have been reported. Furthermore, various symptoms of NQ-induced central nervous disorders were noted in clinical case reports. In particular, the tonic and clonic convulsions induced by NQs in the presence or absence of nonsteroidal anti-inflammatory drugs (NSAIDs) are the most serious disorders (1, 17, 20). Concomitant administration of NSAIDs and NQs is considered an important factor that induces a synergistic interaction that results in convulsions. In order to evaluate the neurotoxic effects induced by the interaction between NQs and NSAIDs, we carried out various in vivo and in vitro experiments. The effect of NSAIDs on the 50% effective dose for an NQ-induced occurrence of convulsions was examined on the basis of in vivo experiments with mice (12). Furthermore, the effects of NQs and/or NSAIDs on in vitro γ-aminobutyric acid type A (GABAA) receptor binding of [3H]GABA and [3H]muscimol was investigated with synaptoneurosomes (7, 28, 29). These in vivo and in vitro experiments indicated that the NQ-induced neurotoxic effect was synergistically increased in the presence of NSAIDs. Recently, we investigated the relationship between the inhibitory effect of NQs on the GABA current response in Xenopus oocytes into which mouse brain mRNA was injected and the molecular structures of NQs. In this study, we could predict quantitatively the intensity of NQ-induced convulsions in vivo from the inhibitory effects of NQs on the GABA response in vitro (12).

However, the quantitative in vivo-in vitro relationship concerning the effects of NQs on the central nervous system in the presence or absence of NSAIDs has not been investigated. In this study, we developed a new toxicodynamic model to evaluate quantitatively the inhibitory effects of both drugs on the GABA current response using GABAA receptor-expressed Xenopus oocytes and obtained various dynamic parameters. Furthermore, these parameters were used to simulate isobolograms for the concentrations of both drugs in the brains in the presence of various inhibitory ratios of the GABAA receptor. We clarified the correlation between those isobolograms simulated in vitro and the isobolograms of the threshold concentration in the brain between the occurrence of convulsions and the lack of occurrence of convulsions caused by both drugs in vivo.

MATERIALS AND METHODS

Materials.

Enoxacin (ENX) and ciprofloxacin (CPFX) were kindly supplied by Dainippon Pharmaceutical Co., Ltd., (Osaka, Japan) and Bayer Pharmaceutical Co., Ltd., (Tokyo, Japan), respectively. Felbinac (FLB) and flurbiprofen (FRP) were kindly supplied by Ledere Japan, Ltd., (Tokyo, Japan) and Kaken Pharmaceutical Co., Ltd., (Chiba, Japan), respectively, and ketoprofen (KTP) was purchased from Sigma Chemical Co. (St. Louis, Mo.). A sodium lauryl sulfate standard was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 131I-labeled human serum albumin (18.5 MBq) was used to estimate the brain tissue capillary volume ratio and was obtained from New England Nuclear (Boston, Mass.). It was used after purification by column chromatography (Sephadex G-25 gel; Pharmacia LKB Biotechnology, Uppsala, Sweden), and the free fraction of iodine was then calculated to be less than 0.3% after ultrafiltration (MPS-3 Centrifree micropartition system Amicon; W. R. Grace and Co., Beverly, Mass.). All other chemicals were commercially available and of analytical grade.

Animals.

Male ddy mice weighing 18 to 20 g were purchased from Seak Yoshitomi (Fukuoka, Japan) and were used after they had been raised in a cage for more than 5 days. Adult female African clawed frogs (Xenopus leavis) were obtained from Hamamatsu Seibutsu Kyozai (Shizuoka, Japan). The Xenopus frogs were kept in an aquarium maintained at about 20°C.

Preparation of injectable solutions.

ENX, FLB, FRP, and KTP were prepared as aqueous solutions of the sodium salt by adding an equimolar amount of NaOH. If the osmotic pressure of these solutions was lower than that of saline, they were made isotonic by adding NaCl. The pH of the injectable solutions used in the experiments was adjusted to neutrality.

Effects of NSAIDs on distribution of ENX in the brain.

ENX (25 mg/kg of body weight) in the presence or absence of FLB (15 mg/kg), KTP (50 mg/kg), or FRP (30 mg/kg) was injected into the tail veins of mice. The total volume of the injectable solutions was 20 μl/g of body weight. The same volume of saline was injected with ENX in the case of the administration of NQs alone. After administration of the drugs, the mice were guillotined at 1, 3, 5, 10, 20, 40, or 60 min, and blood and brain tissue were collected. Sodium heparin was immediately added to the blood samples, and the samples were centrifuged at 1,620 × g for 5 min to obtain the plasma. The plasma and brain tissue were stored in a freezer at −20°C, and their concentrations were analyzed by high-performance liquid chromatography (HPLC).

Convulsion after infusion of ENX.

ENX (3 to 137.5 mg/kg) was administered into the tail veins of mice in the presence or absence of the three NSAIDs (3 to 150 mg/kg), and the occurrence of clonic convulsions within 20 min was observed. In the case of the coadministration of two drugs, it took 1 min to administer each drug, and the NSAIDs were injected at an interval of 1 min after the administration of ENX. Moreover, the same procedure was performed by using each solvent as a control. The mice were guillotined immediately after the occurrence of a convulsion in the first 20 min after drug administration; on the other hand, mice that did not have convulsions were guillotined 20 min after injection of the drugs, and the blood and brain tissue were then collected. Sodium heparin was immediately added to the blood samples, and the samples were centrifuged at 1,620 × g for 5 min to obtain the plasma. The plasma and brain tissue were stored in a freezer at −20°C, and their concentrations were analyzed by HPLC.

Brain tissue and plasma protein binding assay.

The binding of ENX to brain tissue in the presence or absence of NSAIDs (FLB, KTP or FRP) was examined by equilibration dialysis (23). Various concentrations of ENX (14.4, 28.8, and 57.6 μM), FLB (23.6, 47.6, and 94.4 μM), KTP (19.6, 39.2, and 78.6 μM), and FRP (20.0, 40.0, and 80.0 μM) were incorporated into the brain tissue homogenate.

Two milliliters of 5, 10, or 20% brain homogenate containing drugs and 2 ml of 0.1 M phosphate buffer (pH 7.0) were added to both sides of a dialysis membrane (Spectra/Por Membrane; Spectrum Medical Industries, Inc., Laguna Hills, Calif.), and the cells were incubated at 37°C for 10 h. After the incubation, the volumes on both sides of the cells was measured, and 1.0-ml samples were withdrawn. The concentration in the samples were measured by HPLC.

The tissue-unbound fraction was calculated as follows:

graphic file with name M1.gif 1

where fT100 is the predicted tissue-unbound fraction in 100% tissue homogenate, Cf is the concentration of unbound drugs, Cb is the concentration of bound drugs, and D (D = 100/percent homogenate) is the dilution ratio.

The effects of NSAIDs (FLB, KTP, or FRP) on the plasma protein binding and the brain tissue protein binding of ENX were determined by using equilibration dialysis. The concentrations of ENX, FLB, KTP, and FRP were 46.0, 1.93 × 103, 1.57 × 103, and 1.64 × 103 μM, respectively, in the plasma compartment in the dialysis cells. The concentrations of ENX, FLB, KTP, and FRP were 4.6, 47.2, 39.2, and 40.0 μM, respectively, in the brain tissue homogenate compartment in the dialysis cells. These drug concentrations used in the brain tissue and plasma protein binding assays were determined on the basis of the in vivo plasma and brain tissue ENX or NSAID concentration. Two milliliters of 20% of brain homogenate or 0.2 ml of plasma containing drugs and 0.2 ml of 0.1 M phosphate buffer (pH 7.0) was added to both sides of the dialysis membrane (Spectra/Por Membrane; Spectrum Medical Industries, Inc.), and the cells were incubated at 37°C for 10 h. After the incubation, the volumes on both sides of the cells were measured and 1.0 ml of the brain sample or 0.1 ml of the plasma sample was withdrawn. The concentrations in the samples were measured by HPLC.

The plasma-unbound fraction was calculated as follows:

graphic file with name M2.gif

where, fT and fp are the tissue-unbound fraction and the plasma-unbound fraction, respectively, and Cf and Cb are the concentration of unbound drugs and the concentration of bound drugs, respectively. The binding of ENX to the dialysis membrane in the experiments was negligible.

Extraction procedure for determination of ENX and NSAID concentrations in plasma and brain tissue.

The extraction procedure for the determination of the ENX and NSAID concentrations was performed by previously described methods (9, 10). Briefly, in order to extract ENX from plasma samples, 0.9 ml of 0.1 M phosphate buffer, 0.1 ml of 10 μg of CPFX per ml as an internal standard, and 5 ml of chloroform containing 1% ethyl chlorocarbonate (Wako Pure Chemical Industries Ltd.) were added to 0.1 ml of plasma, and the contents were shaken for 10 min and then centrifuged at 1,620 × g for 5 min. After evaporation of the 4-ml organic phase under reduced pressure, the residue was dissolved in 0.1 ml of methanol–0.05 M NaOH (2:1; vol/vol), and 20 μl was injected into the HPLC system. The cerebrum samples were homogenized with a fourfold volume of 0.1 M phosphate buffer. One hundred microliters of 10 μg of CPFX per ml as an internal standard and 5 ml of dichloromethane were added to 1 ml of homogenate. The samples were shaken for 10 min and centrifuged at 1,620 × g for 5 min. Then, 4 ml of 1 mM NaOH was added to 4 ml of the organic phase and the contents were shaken for 10 min. After centrifugation at 1,620 × g for 5 min, 3 ml of the aqueous phase was collected and treated in a manner similar to that described above for the plasma samples, except that 0.1 ml of 10 μg of CPFX per ml was added as an internal standard. The extraction of NSAIDs from the plasma and cerebrum samples was done as described above for ENX. FLB for KTP and FRP and FRP for FLB were used as internal standards.

The limits of quantification were 50 ng/ml for both plasma and brain tissue. For all measurements, coefficients of variation were less than 10%, and within-run accuracies were less than ±10%. Moreover, coefficients of variation for between-day precision were less than 10%.

Correction of concentration in brain tissue.

The equation used for the correction for the concentration in brain tissue was as follows:

graphic file with name M3.gif

where CBR, CBR,obs, R, RB, and Cp represent the real concentration of ENX or NSAIDs in brain tissue, the apparent concentration of ENX or NSAIDs in brain tissue, the cerebral intravascular volume, the ratio of concentration of ENX or NSAIDs in blood to that in plasma, and the concentration of ENX and NSAIDs in plasma, respectively. CBR,obs incoporates the drug remaining in the cerebral intravascular spaces; therefore, CBR was determined by subtracting the amount of drug calculated from both the ratio of the capillary volume occupied in the brain and the concentrations of the drugs in blood from CBR,obs by the equation described above. Consequently, CBR indicates the real drug concentration in the brain tissue. The RB values for ENX, FLB, KTP, and FRP were approximately 1.

HPLC system.

The HPLC system consisted of a liquid chromatograph (Shimadzu LC-9A; Shimadzu, Kyoto, Japan) and a spectrophotometric UV detector (Shimadzu SPD-6AV; Shimadzu) operated at 280 nm. The column was stainless steel (250 by 6 mm [inner diameter]) and was packed with Nucleosil 5C18 (Chemco, Osaka, Japan). The mobile phase was methanol–5 mM sodium dodecyl sulfate, and the pH was adjusted to 2.5 by adding phosphoric acid. The flow rate of the mobile phase was 0.8 ml/min. All analyses were performed at room temperature.

Extraction of mRNA from mouse brain.

The mice were guillotined after dislocation of the neck, and the whole brain (cerebrum, cerebellum, and brain stem) was removed. Total RNA was extracted from the homogenized brain with a phenol-chloroform mixture containing guanidinium thiocyanate, sarcosyl, and mercaptoethanol by the method of Chomczynski and Sacchi (4). Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatography with an mRNA purification kit (Pharmacia LKB Biotechnology, Uppsala, Sweden). The mRNA was stored in a sterile aqueous solution buffer containing 1 mM EDTA and 10 mM Tris-HCl (pH 7.4) at −70°C.

Injection of mRNA into Xenopus oocytes.

Injection of mouse brain mRNA into Xenopus oocytes was performed by the methods described previously (2, 8, 11, 12). Xenopus oocytes were anesthetized with crushed ice, and small pieces of the ovaries were removed surgically. Ovarian oocytes were treated with 1.0 mg of collagenase per ml in modified Barth’s medium containing 88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, and 5 mM Tris-HCl at pH 7.4 for about 30 min at room temperature. After removal of the follicle cell layer, stage V to VI oocytes, which were 1.0 to 1.2 mm in diameter, were selected visually for injection of mRNA. These oocytes had a resting potential of about −40 mV. About 30 ng of mRNA from mouse brain was injected into each oocyte with a glass pipette with a tip diameter of 10 μm. The oocytes into which mRNA was injected were kept in a broth of modified Barth’s medium at 20°C until they were used for electrophysiological measurements.

Dose dependency of effect of NSAIDs on ENX-induced inhibition of GABA current response.

We measured the enhanced inhibitory effects on the GABA current response in the presence of NSAIDs by the method of Kawakami et al. (12). Briefly, the concentration dependency of the ENX-induced alteration of the GABA current response was measured in the presence of 1, 10, or 100 μM NSAIDs or in the absence of NSAIDs.

Electrophysiological measurement of GABAA receptor response.

The current response of Xenopus oocytes into which mouse brain mRNA was injected was obtained by a previously described method (8). Glass microelectrodes were placed into the oocytes, and the membrane potential was maintained at −60 mV. A microscopic electrode differential amplifier (DPZ-16; Daia Medical System Ltd., Tokyo, Japan) and a membrane-fixed amplifier (CEZ-1100; Kouden Industries Japan, Ltd., Tokyo, Japan) and a membrane-fixed amplifier (CEZ-100; Kouden Industries Japan, Ltd., Tokyo, Japan) were used to measure the current response.

Effects of NSAIDs on transcellular transport in MBEC4s.

Cultured mouse brain capillary endothelial cells (MBEC4s) were used as described previously (15, 27). In brief, MBEC4 was maintained in Dulbecco’s modified Eagle’s medium (Nikken Bio Medical Laboratory, Kyoto, Japan) containing 10% fetal calf serum, 100 U of penicillin per ml, and 100 μg of streptomycin per ml. In the transport study, the number of cells was diluted to 4 × 104 cells/ml, and the cells were examined with a Transwell apparatus (Costar, Cambridge, Mass.). One milliliter of the cell suspension was seeded in the cup, cultured in 5% CO2–95% air for 3 days, and washed three times with buffer (141 mM NaCl, 4.0 mM KCl, 2.8 mM CaCl2, 1.0 mM MgSO4, 10 mM d-glucose, 10 mM HEPES [pH 7.4]) at 37°C. MBEC4s possess the same fundamental properties as brain capillary endothelial cells. The objective of the transport experiments with MBEC4s is to confirm whether the potentiated ENX-induced convulsions in the presence of NSAIDs was not due to the enhanced permeation of ENX across the blood-brain barrier by NSAIDs.

The cups were placed in a dish, and 37°C buffer was placed in the albuminal side. Immediately, 37°C medium containing ENX at 2.16 × 10−2 μM in the presence of NSAIDs (11.8 μM FLB, 17.7 μM KTP, 18.4 μM FRP) or in the absence of NSAIDs was put in the luminal side and the assay was begun. A 0.5-ml sample was collected from the abluminal side at every sampling time, and the same amount of 37°C buffer was added to the abluminal side to maintain the volume. The concentration in each sample was measured by HPLC.

Toxicodynamic analysis.

We constructed a newly developed dynamic model that considers the receptor binding and dissociation to evaluate the effect of ENX and/or NSAIDs on the GABA current response in GABAA receptors. Mass balance equations for receptor binding and dissociation of ENX and NSAIDs were as follows.

graphic file with name M4.gif 2
graphic file with name M5.gif 3
graphic file with name M6.gif 4

where, [Q], [R], and [S] are the unbound concentration of ENX, the concentration of free GABA receptors, and the unbound concentration of NSAIDs, respectively; [QR], [SR], and [QSR] are the concentrations of the complex of Q and R, S and R, and Q, S, and R, respectively; Ki is the inhibitory constant of ENX for the GABAA receptor; Kd is the dissociation constant of NSAIDs for the GABAA receptor; Ki′ is the dissociation constant between the NSAID-receptor complex and ENX; and γ is a hill coefficient (the number of NSAIDs) concerning the interaction between NSAIDs and the GABAA receptor. Moreover, it is assumed that the binding affinity to ENX increases in the presence of NSAIDs; that is, Ki is greater than Ki′. The total number of receptors is [R0] and is expressed as follows.

graphic file with name M7.gif 5

Furthermore, the receptor occupancy (φ), that is, the inhibition ratio relative to that for the GABAA receptor, is as follows:

graphic file with name M8.gif 6

Furthermore, φ is expressed as follows by using various dynamic parameters and equations 1 to 5:

graphic file with name M9.gif 7
graphic file with name M10.gif

Therefore, the GABA response rate (R) is as follows.

graphic file with name M11.gif
graphic file with name M12.gif
graphic file with name M13.gif 8

The dynamic parameters Ki, Ki, and Kdγ were determined by the fitting of the GABA response (R) profile in the presence of various concentrations of NQs and/or NSAIDs to equation 8 by the nonlinear least-squares method (MULTI) (32). All data were weighed by the reciprocal of the square of observed values for nonlinear least-squares regression. The optimum receptor occupancy, φ, was determined by eye-fitting within the φ range of from 0.01 to 0.1 at an interval of 0.01, which most exactly represents the observed values. In addition, equation 9 was used to construct an isobologram (6, 13, 24–26) based on the relationship between [Q] and [S], as follows:

graphic file with name M14.gif
graphic file with name M15.gif 9

The total concentrations in brain tissue ([s] and [q]) are the calculated brain unbound concentrations ([S] and [Q]) and the unbound fraction in the brain (fT100s for NSAIDs, fT100q for ENX).

graphic file with name M16.gif 10
graphic file with name M17.gif 11

The simulation of the isobologram based on the relationship between [s] and [q] by substituting Ki, Ki, Kdγ, fT100s, and fT100q in equations 9, 10, and 11 was carried out.

RESULTS

In vivo experiments.

Figure 1 shows the isobolograms based on the relationship between the occurrence of convulsion and the concentrations of ENX and the three NSAIDs in brain tissue. Remarkable threshold lines were obtained for mice with convulsions and those without convulsions, and the lines had concave patterns. We confirmed that the interaction between ENX and the three NSAIDs was the synergistic interaction by the analysis of isobolograms.

FIG. 1.

FIG. 1

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Isobologram based on ENX-induced convulsions in the presence of FLB (A), KTP (B), or FRP (C), ENX (3 to 137.5 mg/kg) was injected into the tail veins of mice in the presence of NSAIDs (3 to 150 mg/kg). The occurrence of clonic convulsion within 20 min after administration of drugs was observed, and the brain was collected. The concentration of the drugs in brain tissue was determined by HPLC as described in Materials and Methods. Symbols: ○, mice without convulsions; ●, mice with convulsions.

Figure 2 indicates the brain and plasma concentration-time profiles for ENX in the presence or absence of the three NSAIDs (FLB, KTP, and FRP). Differences in the concentrations of ENX in plasma and brain tissue were not observed in the presence of the three NSAIDs.

FIG. 2.

FIG. 2

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Effect of the NSAID FLB (A), KTP (B), or FRP (C) on plasma and brain concentration-time profiles for ENX. ENX (25 mg/kg) was injected into the tail veins of the mice in the presence or absence of NSAIDs (FLB, 15 mg/kg; KTP, 50 mg/kg; FRP, 30 mg/kg). The mice were guillotined at 1, 3, 5, 10, 20, 40, or 60 min after administration of the drugs, and the plasma and brain were collected. Plasma and brain tissue ENX concentrations were measured by HPLC. Each value represents the mean ± SD (n = 5). Symbols; □, plasma ENX concentration in the absence of NSAIDs; ■, plasma ENX concentration in the presence of NSAIDs; ○, brain tissue ENX concentration in the absence of NSAIDs; ●, brain tissue ENX concentration in the presence of NSAIDs.

In vitro experiments.

Figure 3 shows the ENX-induced inhibition of the GABA current response in the presence of NSAIDs (1, 10, and 100 μM) or the absence of NSAIDs in Xenopus oocytes into which mouse brain mRNA was injected. In the presence of NSAIDs, the GABA response was inhibited in a dose-dependent manner. Moreover, the concentration-response curves of ENX shifted leftward in the presence of the three NSAIDs. No effect on the GABA response in the presence of NSAIDs alone (1 to 100 μM) was observed. The dynamic parameters of ENX calculated by using the dynamic model in the presence of the NSAIDs are presented in Table 1. The order of inhibition (Ki′) in the presence of ENX and NSAIDs was FLB (0.2 μM) < FRP (0.3 μM) < KTP (1.2 μM). The inhibitory potencies determined by Ki/Ki′ were approximately 280, 180, and 50 for ENX-FLB, ENX-FRP, and ENX-KTP, respectively.

FIG. 3.

FIG. 3

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Inhibitory effect of the NSAID FLB (A), KTP (B), or FRP (C) on GABA response to ENX. The inhibitory effect on ENX on the GABA (10 μM)-induced current response in the presence (1 [●], 10 [▴], and 100 μM [■] or absence (○) of NSAIDs was investigated with Xenopus oocytes into which mouse brain mRNA was injected, as described in Materials and Methods. The solid lines in the figure were obtained by fitting the GABA-induced response to equation 8 by using MULTI, as described in Materials and Methods.

TABLE 1.

Dynamic parameters for ENX-induced inhibition of GABA-induced response in the presence of FLB, FRP, or KTPa

Parameter ENX-FLB ENX-FRP ENX-KTP
Ki (μM) 56.6 ± 10.5 56.6 ± 9.66 56.8 ± 10.6
Ki (μM) 0.199 ± 0.0423 0.311 ± 0.0554 1.19 ± 0.0424
Kdγ (μM) 69.1 ± 21.1 69.0 ± 21.2 77.1 ± 20.8
γ 1.52 ± 0.166 1.66 ± 6.17 1.79 ± 0.167
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a

The inhibitory effect of ENX on GABA-induced response in the presence of NSAIDs was fit by the nonlinear least-squares method (MULTI) described in Materials and Methods, and the dynamic parameters (Ki, Ki, Kdγ, and γ) were calculated. Each value represents the mean ± SD (n = 3). Ki is the inhibitory constant of ENX for GABAA receptor, Kd is the dissociation constant of NSAIDs for the GABAA receptor, Ki is the discussion constant between the NSAID-receptor complex and ENX, and γ is a hill coefficient (the number of NSAIDs) concerning the interaction between NSAIDs and the GABAA receptor. 

The brain tissue-unbound fraction (fT) of ENX was measured. ENX showed relatively low levels of binding (50.2% ± 15.9%, mean ± standard deviation [SD]; n = 6), while FLB, FRP, and KTP showed relatively high levels of binding (5.6% ± 2.8%, 9.2% ± 4.5%, and 30.9% ± 20.7%, respectively; mean ± SD; n = 6). Moreover, the effects of NSAIDs on the binding of ENX to plasma and brain tissue was examined. The levels of binding of ENX to plasma and brain tissue were 69.1% ± 2.4% and 51.7% ± 3.1% (mean ± SD; n = 3), respectively, while there was no alteration in the binding of ENX to plasma in the presence of FLB, KTP, and FRP (71.2% ± 2.7%, 69.9% ± 2.6%, and 68.7% ± 2.7%, respectively; mean ± SD; n = 3). No alteration in the binding of ENX to the brain tissue in the presence of FLB, KTP, and FRP was observed (51.3% ± 3.6%, 51.3% ± 4.3%, and 52.4% ± 3.1%, respectively; mean ± SD; n = 3).

The effects of NSAIDs on the transcellular transport of ENX in MBEC4s is shown in Fig. 4. There was no change in the transcellular transport rate of ENX from the luminal side to the abluminal side in the presence of the three NSAIDs.

FIG. 4.

FIG. 4

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Effect of the NSAID FLB (A), KTP (B), or FRP (C) on transcellular transport of ENX across MBEC4 from the luminal to the abluminal side. The transcellular transport of ENX in the presence or absence of NSAIDs was determined as described in Materials and Methods. Each sample was collected at the scheduled time, and the drug concentration was measured by HPLC. Each value represents the mean ± SD (n = 4). Symbols: ○, control; ●, effect in the presence of FLB; ▴, effect in the presence of KTP; ■, effect in the presence of FRP.

Correlation between in vivo and in vitro data.

Figure 5 shows the simulation lines of the GABA response based on the isobolograms for the concentrations of ENX and NSAIDs in brain tissue according to equation 9. These were calculated by using Ki, Ki′, Kdγ, γ, fT100s, and fT100q. Various simulation lines were fitted to the isobolograms (Fig. 1) for the observed threshold concentration in brain tissue between mice with convulsions and those without convulsions, and φ was estimated. The estimated φ values were 0.02 for ENX-FRP, 0.02 for ENX-KTP, and 0.01 for ENX-FLB, suggesting that there is no difference in φ among the three NSAIDs.

FIG. 5.

FIG. 5

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Simulation of isobologram for convulsion induced by ENX in the presence of the NSAID FLB (A), KTP (B), FRP (C). The isobologram was simulated according to equations 9, 10, and 11 as described in Materials and Methods. Solid lines are simulation lines. The line nearest the origin represents 0.01 of the GABA-induced current response (f), and simulation was carried out within the range of 0.01 to 0.1 of f at equal intervals of 0.01. Lines were generated at equal intervals. The hatched zone represents the optimum area of the threshold concentration in brain tissue for the occurrence of convulsions by the interaction between ENX and NSAIDs.

DISCUSSION

The threshold lines for convulsions and nonconvulsions after the administration of ENX in the presence or absence of NSAIDs in isobolograms based on the concentrations of both drugs in brain tissue showed concave patterns, suggesting a synergistic effect on ENX-induced convulsions in the presence of NSAIDs (Fig. 1). Plasma and brain concentration-time profiles for ENX, the transport rate of ENX across the blood-brain barrier, and fp and fT of ENX did not change in the presence of the NSAIDs as, shown in Fig. 2 and Fig. 4, indicating that these synergistic effects were not due to the pharmacokinetic interaction between ENX and NSAIDs. In the Xenopus oocytes into which mouse brain mRNA was injected, the GABA response was inhibited by ENX in a concentration-dependent manner, and the concentration-response curves of ENX were shifted leftward in the presence of the NSAIDs (Fig. 3). These findings were consistent with the relationship between FLB and CPFX, norfloxacin, or ofloxacin (12). Furthermore, our previous report indicated that the effect of ENX on the GABA response in vitro was synergistically potentiated in the same manner with the in vivo synergistic interaction on ENX-induced convulsions in the presence of the NSAIDs (11). In this study, based on an analysis of concentration-response curves by a newly developed toxicodynamic model, we reproduced quantitatively the in vivo synergistic interaction and obtained the dynamic parameters (Table 1). The order of inhibition (Ki) for ENX on the GABA response in the presence of the NSAIDs was FLB (0.2 μM) < FRP (0.3 μM) < KTP (1.2 μM), and the inhibitory effect of ENX alone was 50- to 280-fold lower than that of ENX in the presence of the NSAIDs. We simulated the isobologram for the concentration of both drugs in brain tissue by using the dynamic parameters (Table 1) and the brain tissue-unbound fraction of both drugs by using equation 9.

We made up 10 simulation lines within the range of 0.01 to 0.1 as φ, and the lines obtained fit the isobologram for the threshold concentration in brain tissue between mice with convulsions and those without convulsions in vivo. The φ range of 0.01 to 0.02, despite the kind of NSAID, that was obtained indicated that 1 to 2% or more blockade of the GABAA receptor was necessary to induce convulsions on the basis of the inhibition of the GABA response.

In conclusion, in order to predict quantitatively the toxicodynamic interaction between ENX and NSAIDs and to estimate the isobolograms of the threshold concentration in brain tissue between mice with convulsions and mice without convulsions, kinetic analysis based on the toxicodynamic parameters (Ki, Ki′, Kdγ, γ, and φ values [0.01 to 0.02]) and the pharmacokinetic parameters (fT and fp) are useful.

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