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Drug Metab. Pharmacokinet. 24 (2): 167–174 (2009). Regular Article Quantitative Comparison of the Convulsive activity of Combinations of Twelve Fluoroquinolones with Five Nonsteroidal Antiinflammatory Agents

Jahye KIM1, Hisakazu OHTANI2, Masayuki TSUJIMOTO1,† and Yasufumi SAWADA2,3,* 1Department of Medico-Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 2Laboratory of Drug Informatics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan 3Graduate School of Interdisciplinary Information Studies, The University of Tokyo, Tokyo, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: Concomitant administration of certain fluoroquinolone antimicrobials and nonsteroidal antiin- flammatory agents (NSAIDs) induces serious convulsion in humans. There are differences in convulsive activ- ity among fluoroquinolones and in the potentiation of fluoroquinolone-induced convulsion among NSAIDs, but a comprehensive, quantitative comparison has not been carried out. This study evaluates the inhibitory ef- fects of twelve fluoroquinolones (, , , , , , , , pazufloxacin, , , and ) alone or in the presence of

an NSAID (4-biphenylacetic acid, sodium, , or zaltoprofen) on the GABAA receptor binding of [3H]muscimol in an in vitro study using mice synaptic plasma membrane. The rank order of inhibitory effects of the fluoroquinolones was prulifloxacin§norfloxacinÀciprofloxacinÆenoxacinÀgati- floxacinÆofloxacin§tosufloxacin§lomefloxacinÀlevofloxacinÆsparfloxacinÆpazufloxacin§fleroxacin. 4- Biphenylacetic acid most potently enhanced the inhibitory effects of the fluoroquinolones, while zaltoprofen, loxoprofen, lornoxicam and diclofenac had essentially no effect. The clinical risk of convulsion for each com- bination was estimated using a pharmacodynamic model based on receptor occupancy using the in vitro data set obtained and pharmacokinetic parameters in humans collected from the literature. The combinations of 4- biphenylacetic acid with prulifloxacin and enoxacin were concluded to be the most hazardous.

Keywords: Fluoroquinolone; NSAIDs; GABAA inhibition; Drug interaction; Pharmacodynamic model- ing

fluoroquinolones.1) Fluoroquinolone-induced CNS excita- Introduction tion is attributable to the inhibition of g-aminobutyric 3) Fluoroquinolone antimicrobial agents are widely used acid (GABA) binding to the GABAA receptor. Some for the treatment of various infections, because of their NSAIDs such as , potentiate the blockade of the 1) broad spectrum of antimicrobial activity. Central ner- GABAA receptor by fluoroquinolones, though fenbufen 4) vous system (CNS) reactions such as headache, dizziness, itself does not inhibit GABAA receptor binding at all. convulsion, insomnia and psychosis are clinically sig- Previous reports suggest that there are differences ac- nificant side effects of these agents, although their inci- cording to the fluoroquinolone in convulsive activity and dence is as low as 1–2%.2) Severe CNS adverse reactions differences among NSAIDs in the potentiation of of fluoroquinolones include convulsions, and the inci- fluoroquinolone-induced convulsions,5) but comprehen- dence of convulsion increases when nonsteroidal antiin- sive information is not yet available. We previously flammatory agents (NSAIDs) are used concomitantly with reported that the convulsive activity of at least some

Received: May 16, 2008, Accepted; November 12, 2008 *To whom correspondence should be addressed: Yasufumi SAWADA, Laboratory of Drug Informatics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. Tel. ＋81-3-5841-2270, Fax. ＋81-3-5802-1570, Email: sawada＠mol.f.u-tokyo.ac.jp †Present address: Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kyoto Pharmaceutical University, Kyoto, Japan

167 168 Jahye KIM, et al. combinations of fluoroquinolones and NSAIDs is syner- and the supernatant was centrifuged at 20,000×gfor20 gistic, as determined using isobolograms for the occur- min. The crude pellet was suspended in 50 volumes 50 rence of convulsions and the concentrations of enoxacin mM Tris-HCl buffer (pH 7.4) using a sonicator (Vibtra- (ENX) and three NSAIDs in mouse brain.6) We have also Cell VCX400, Sonics and Materials, CT, USA) and cen- shown that the convulsive activity of fluoroquinolones in trifuged at 48,000×g for 20 min. The resultant pellet vivo can be estimated from in vitro studies of receptor oc- was suspended in 0.05% Triton X-100, incubated at 379 cupancy.6,7) C for 20 min and washed three times in the buffer. All This study investigates the convulsive activity of twelve these processes were carried out at 49C, except for fluoroquinolones in the presence or absence of five 0.05% Triton X-100 treatment. The final suspension NSAIDs, using GABAA receptor binding assay. For fen- couldbestoredat－809C for at least 90 days without bufen, we investigated the effect of its metabolite, 4- loss of binding capacity. biphenylacetic acid (BPAA), which accounts for the Inhibitory effects of fluoroquinolones and potentiation of convulsive activity.6) We also estimated NSAIDs on [3H]muscimol binding: The inhibitory the risk of convulsion in a clinical setting using a phar- effects of 60 combinations of 12 fluoroquinolones macodynamic model based on receptor occupancy. (CPFX, ENX, FLRX, GFLX, LVFX, LFLX, NFLX, OFLX, PZFX, PUFX; NM394, SPFX, TFLX) and 5 NSAIDs Methods (BPAA, diclofenac sodium, loxoprofen, lornoxicam, zal- 3 Materials: Pazufloxacin (PZFX) and tosufloxacin toprofen) on GABAA receptor were analyzed by [ H]mus- (TFLX) were kindly supplied by Mitsubishi Pharma Co., cimol binding assay.8) To clarify the potentiating effects Ltd. (Tokyo, Japan) and Taisho Toyama Pharmaceutical of NSAIDs, unlabeled quinolones (solubilityÃ300 mM) Co., Ltd. (Tokyo, Japan), respectively. The active metabo- and [3H]muscimol were incubated with membrane sus- lite of prulifloxacin (PUFX), NM394, was supplied by pension in the absence or presence of 1, 10, or 100 mM MeijiSeikaKaisha,Ltd.(Tokyo,Japan).Enoxacin(ENX), NSAIDs. The binding preparation (1 mL), consisting of ofloxacin (OFLX), lomefloxacin (LFLX) and norfloxacin 100 mL membrane suspension (final 3.0 mg protein/mL), (NFLX) were purchased from Sigma Chemical Co., Ltd. 200 mL[3H]muscimol (10 nM) solution, 100 mLbufferor (St. Louis, MO, USA). Ciprofloxacin (CFLX), fleroxacin quinolone solution and 100 mLbufferorNSAIDssolu- (FLRX), gatifloxacin (GFLX), levofloxacin (LVFX) and tion, was incubated at 49C for 30 min. An aliquot of 800 sparfloxacin (SPFX) were purchased from Wako Pure mL of the preparation was filtered through a glass fiber Chemical Industries, Ltd. (Osaka, Japan). Lornoxicam filter (GF/C, Whatman Inc., Clifton, NJ, USA) and and zaltoprofen were kindly supplied by Taisho Toyama washed twice with 5 mL ice-cold buffer using a cell har- Pharmaceutical Co., Ltd. and Nippon Chemiphar Co., vester (Millipore Intertech, MA, USA). The radioactivity Ltd. (Tokyo, Japan), respectively. Loxoprofen, 4- of the filters was determined in 5 mL scintillation cock- biphenylacetic acid (BPAA; the active metabolite of fen- tail (Clearsol I, Nacalai Tesque, Kyoto, Japan), using a liq- bufen) and diclofenac sodium were purchased from Sig- uid scintillation counter (LC3500, Aloka Co. Ltd., Tokyo, ma Chemical Co., Ltd. and Wako Pure Chemical Indus- Japan). Nonspecific binding was determined in the tries, Ltd. [3H]Muscimol (specific activity; 29.5 Ci/mmol), presence of 1 mM unlabled GABA and the results were a specific GABAA receptor agonist, was purchased from expressed as specific [3H]muscimol binding (percent of Perkin Elmer Life Sciences, Inc. (Boston, MA, USA). All control). other reagents were commercial products of analytical Pharmacodynamic model: To analyze the inhibi- grade. tory effects of the combinations of the fluoroquinolones 3 Animals: The protocol for the animal study was ap- and NSAIDs on [ H]muscimol binding to the GABAA proved by the Ethical Committee on Animal Experiments receptor, the following model, previously developed by of the Faculty of Pharmaceutical Sciences, Kyushu Uni- us,6) wasusedwithminormodification.Withregardto versity. Male ddY mice (18–20 g) were purchased from the binding of fluoroquinolones and NSAIDs to GABAA Seak Yoshitomi (Fukuoka, Japan) and kept under stan- receptor, three mass balance equations1–3) may be writ- dard laboratory conditions (food and water ad libitum, ten, as room temperature 22±19C and light on from 7:00 to Ki＝[Q]･[R]/[QR] (1) 19:00) for at least 7 days before use. Kdg＝[S]g･[R]/[SgR] (2) Preparation of synaptic plasma membranes: Ki?＝[Q]･[SgR]/[QSgR] (3) Synaptic plasma membranes were prepared from the brains of ddY mice by the method of Enna et al.,8) with where [Q], [S] and [R] represent the free concentrations minor modification. Whole brains were homogenized in of fluoroquinolone, NSAID and GABAA receptor, respec- 10 volumes 0.32 M sucrose using a Teflon-on-glass tissue tively. [QR], [SgR], and [QSgR] represent the concentra- homogenizer (Mini D.C. stirrer, EYLA, Tokyo, Japan). tions of respective complexes. Ki (mM) is the inhibitory The homogenate was centrifuged at 1,000×gfor10min constant of the fluoroquinolone for the GABAA receptor, Convulsions by Fluoroquinolones and NSAIDs 169

Ki? (mM) is the inhibitory constant of the fluoroquinolone mL/g tissue, unless otherwise stated in the literature and for the NSAID-GABAA receptor complex, Kd (mM) is the used to correct Kp. The unbound fraction of each drug in dissociation constant of the NSAID for GABAA receptor the whole brain (fT100) was also assumed equal to that in and g is the Hill coefficient (number of NSAID molecules mice, measured by equilibrium dialysis and HPLC as 6) that interact with the GABAA receptor). We assumed that previously described. NSAID increases the binding affinity of fluoroquinolones Prediction of GABAA receptor occupancy of (KiÀKi?). The receptor occupancy (F) of the fluoro- fluoroquinolones in the human brain: Putative quinolones is described by equation (4). GABAA receptor occupancy (Fpred) of a fluoroquinolone in the human brain was estimated by substituting [Q] and F＝([QR]＋[QSgR])/[Rtot](4) [S] into equation (5) with Cbrain, f of the fluoroquinolone where [Rtot] represents the total concentration of the and NSAID, respectively, derived from equation (7). GABA receptor, i.e., the sum of [R], [QR], [SgR] and A Results [QSgR]. Substitution of equations (1)–(3) into equation (4) Inhibitory effects of fluoroquinolones and yields equation (5). NSAIDs on [3H]muscimol binding: [3H]Muscimol bound to synapse membranes both concentration- and in- F＝[Q]･(Ki?･Kdg＋Ki･[S]g)/sKi?･Kdg･([Q]＋Ki) cubation time-dependently (data not shown). The binding ＋(Ki?＋[Q])･[S]g･Kit (5) affinity (Kd) and maximal binding capacity (Bmax)of Then, specific binding of [3H]muscimol (SB; percent of [3H]muscimol were 41.5±3.50 nM and 6.78±0.327 control) may be represented by equation (6). pmol/mg protein (mean±S.E.M.), respectively. Figure 1 shows the inhibition curves of the 12 fluoroquinolones in SB(%)＝100･(1－F) the absence or presence of 1, 10 or 100 mMBPAAandKi ＝100･(Ki･Ki?･Kdg＋Ki･Ki?･[S]g) of fluoroquinolones are listed in Table 1.Evenintheab- ×sKi?･Kdg･([Q]＋Ki) sence of BPAA, all the fluoroquinolones inhibited ＋(Ki?＋[Q])･[S]g･Kit (6) [3H]muscimol binding in a concentration-dependent Equation (6) was simultaneously fitted to the inhibitory manner. The inhibition was potentiated by BPAA in a response curves of each fluoroquinolone in the presence concentration-dependent manner. Inhibition curves of and absence of an NSAID by a nonlinear least-squares the 12 fluoroquinolones in the absence and presence of regression using Marquardt-Levenberg iterative curve-fit- 1, 10 and 100 mM zaltoprofen, loxoprofen, lornoxicam ting method (MLAB, Civilized Software, MD, USA) with and diclofenac sodium were also obtained (Figure not constant weight to obtain Ki and Ki/Ki? for each combi- shown). nation of fluoroquinolone and NSAID and Kdg and g for Analysis based on the pharmacodynamic model: each NSAID. The model described in the methods section was fitted to Estimation of unbound concentrations of drugs each combination of a fluoroquinolone and NSAID to ob- in the human brain: To quantitatively estimate the tain parameters. By the curve-fittings (as shown in Fig. risk of convulsion in humans, we estimated the unbound 1), obtained pharmacodynamic parameters for the 60 concentrations of drugs in the human brain (Cbrain, f)with combinations of 12 fluoroquinolones (CPFX, ENX, equation (7). FLRX, GFLX, LVFX, LFLX, NFLX, OFLX, PZFX, PUFX; NM394, SPFX, TFLX) and 5 NSAIDs (BPAA, diclofenac C ＝C ･K ･f (7) brain, f ss (max) p T100 sodium, loxoprofen, lornoxicam, zaltoprofen) are listed where Css (max),Kp and fT100 represent the maximum plas- in Tables 1–5. The rank order of inhibitory potencies of ma concentration of drug at the steady state after the the fluoroquinolones (Ki) was PUFX§NFLXÀCPFXÆ standard dosage regimen, the ratio of AUCbrain to ENXÀGFLXÆOFLX§TFLX§LFLXÀLVFXÆSPFXÆ AUCplasma in rats or mice and the unbound fraction of PZFX§FLRX. The order of potentiation of 5 NSAIDs each drug in the whole brain, respectively. First, the plas- for inhibition by fluoroquinolones was BPAAÀÀzalto- ma concentration profile of each drug after single oral profenÀloxoprofenÀlornoxicamÆdiclofenac, as shown administration was obtained from the literature and 1- or in Figure 2.Ingeneral,PUFX,NFLX,ENXandCPFX 2-compartment open model with first order absorption show relatively larger Ki/Ki? than others irrespective of was fitted to the profile by a nonlinear least-squares NSAIDs, implying these fluoroquinolones are susceptible regression method9) to obtain respective pharmacokinetic to potentiation of convulsive activity by NSAIDs. parameters. Css (max) was calculated from the paramenters. Prediction of GABAA receptor occupancies of Kp of each drug in humans was assumed equal to that in fluoroquinolones in the human brain: Table 6 rats or mice, as indicated in the literature. Kp was calcu- shows Cbrain, f of each drug obtained using Kp,fT100 and lated as the ratio of AUCbrain to AUCplasma in rats or mice. Css (max). Table 7 shows Fpred of fluoroquinolones in the Thevolumeofbraincapillarywasassumedtobe0.008 absence or presence of NSAIDs estimated using Cbrain, f 170 Jahye KIM, et al.

Fig. 1. Inhibitory effects of the fluoroquinolones on [3H]muscimol binding to synaptic plasma membrane of mouse brain, in the absence or presence of BPAA Various concentrations of unlabeled fluoroquinolones and [3H]muscimol (10 nM) were incubated with synaptic plasma membrane for 30 min at 49C, in the absence (open circle) or presence of 1 mM (closed circle), 10 mM (open square), or 100 mM (closed square) BPAA. Equation (6) was fitted to the data by non-linear least square method and best-fit curves are presented as solid lines (n＝3). Error bars are omitted for clarity.

Table 1. Pharmacodynamic parameters for inhibition of the Table 2. Pharmacodynamic parameters for inhibition of the

GABAA receptor induced by fluoroquinolones in the presence GABAA receptor induced by fluoroquinolones in the presence of BPAA of zaltoprofen

NQs Ki (mM) Ki? (mM) Ki/Ki? (63% CI) NQs Ki (mM) Ki? (mM) Ki/Ki? (63% CI)

NFLX 39.2±12.7 0.00144 27200 (7880¿94000) NFLX 49.0±11.2 0.203 241 (20.1¿2890) PUFX 39.9±12.9 0.0000594 672000 (196000¿2310000) PUFX 40.6±8.4 0.397 102 (8.86¿1180) CPFX 105±34 0.0124 8475 (2400¿29900) CPFX 117±30 0.205 572 (47.5¿6890) ENX 210±73 0.000631 333000 (98000¿1130000) ENX 197±54 0.080 2450 (209.7¿28500) GFLX 572±187 2.11 271 (77¿957) GFLX 469±100 76.9 6.10 (0.76¿49.0) OFLX 324±94 0.586 553 (156¿1960) OFLX 568±141 18.1 31.4 (2.96¿333) TFLX 419±177 36.1 12 (3.21¿42.1) TFLX 566±245 137 4.13 (0.39¿44.2) LFLX 268±78 0.412 650 (183¿2310) LFLX 599±156 16.5 36.4 (3.39¿391) LVFX 597±194 4.08 147 (42.4¿507) LVFX 898±264 56.4 15.9 (1.59¿159) SPFX 1029±463 2.32 444 (120¿1630) SPFX 1107±378 40.3 27.5 (2.59¿292) PZFX 979±385 11.0 89 (25.3¿313) PZFX 2056±1007 2050 1.00 (0.00433¿233) FLRX 1121±440 60.2 19 (5.35¿64.7) FLRX 2089±1049 257 8.12 (0.750¿87.9)

The inhibitory effects of fluoroquinolones on [3H]muscimol binding in the Further details as in Table 1.Kdg and g for zaltoprofen were estimated as 438 presence of BPAA was fitted to equation (6), and pharmacodynamic ±888 and 0.959±0.151, respectively. parameters were obtained as described in ``Materials and Method''. Each value represents the estimate±SD (n＝3). Ki is the inhibitory constant of fluoro- quinolones for GABAA receptor, Ki? is the inhibitory constant of fluoroquino- g lones for NSAIDs-GABAA receptor complex, Kd is the dissociation constant of NSAIDs for GABAA receptor and g is the number of NSAID molecules that in- g teract with GABAA receptor. Kd and g for BPAA were estimated as 2700± 2660 and 1.45±0.11, respectively. Convulsions by Fluoroquinolones and NSAIDs 171

Table 3. Pharmacodynamic parameters for inhibition of the

GABAA receptor induced by fluoroquinolones in the presence of loxoprofe

NQs Ki (mM) Ki? (mM) Ki/Ki? (63% CI)

NFLX 34.2±4.5 16.8 2.04 (1.66¿2.52) PUFX 53.9±7.2 26.0 2.08 (1.69¿2.55) CPFX 99±16 9.83 10.1 (7.98¿12.7) ENX 260±50 8.68 30.0 (26.1¿42.7) GFLX 540±96 372 1.45 (1.11¿1.89) OFLX 546±98 408 1.34 (1.02¿1.76) TFLX 563±193 493 1.14 (0.673¿1.94) LFLX 692±140 340 2.04 (1.54¿2.69) LVFX 1168±312 651 1.80 (1.25¿2.58) SPFX 1403±424 652 2.15 (1.45¿3.19) Fig. 2. Potentiation of the inhibitory effects of ENX on PZFX 1931±722 2130 0.908 (0.482¿1.71) 3 FLRX 2100±838 1962 1.07 (0.571¿2.01) [ H]muscimol binding by five NSAIDs Various concentrations of unlabled ENX and [3H]muscimol (10 nM) Further details as in Table 1.TheKdg and g values for loxoprofen were esti- were incubated with synaptic plasma membrane for 30 min at 49C, mated to be 12.5±3.7 and 1.54±0.26, respectively. inthepresenceof100mMNSAID(n＝3). Error bars are omitted for clarity.

Table 4. Pharmacodynamic parameters for inhibition of the (Table 6) and pharmacodynamic parameters (Tables GABAA receptor induced by fluoroquinolones in the presence of lornoxicam 1–5). Zaltoprofen, loxoprofen, lornoxicam and diclofenac sodium were considered unlikely to increase NQs Ki (mM) Ki? (mM) Ki/Ki? (63% CI) Fpred of fluoroquinolones. NFLX 46.1±4.8 12.4 3.73 (0.051¿271) PUFX 56.3±5.2 62.8 0.90 (0.420¿1.92) Discussion CPFX 158±18 36.37 4.3 (0.045¿421) This study estimates the risk of convulsion in humans ENX 176±25 17.61 10.0 (0.042¿1220) GFLX 510±65 237 2.15 (0.093¿49.3) following concomitant administration of fluoroquino- OFLX 515±67 227 2.27 (0.085¿60.7) loneswithorwithoutNSAIDsonthebasisofphar- TFLX 556±137 487 1.14 (0.390¿3.34) macokinetic and pharmacodynamic analyses. The phar- LFLX 589±81 206 2.85 (0.063¿129) macodynamic parameters for the binding of fluoroquino- LVFX 1095±201 683 1.60 (0.171¿15.1) lones and NSAIDs were obtained by the in vitro study in SPFX 1140±214 742 1.54 (0.193¿12.2) PZFX 1982±548 1940 1.02 (0.382¿2.73) mice, while their pharmacokinetic parameters in humans FLRX 2107±611 1520 1.39 (0.226¿8.50) were taken from the literature. The present model-based approach enables us to estimate quantitatively the risk of Further details as in Table 1.Kdg and g for lornoxicam were estimated as 201 ±291 and 1.18±3.04, respectively. convulsion of fluoroquinolones and potentiation by NSAIDs in humans from in vitro data and pharmacokinet- ic parameters. This approach becomes practical only Table 5. Pharmacodynamic parameters for inhibition of the when comprehensive in vitro data on the inhibitory ef- GABAA receptor induced by fluoroquinolones in the presence fects of various fluoroquinolones in the presence of vari- of diclofenac sodium ous NSAIDs are available and therefore, we measured a NQs Ki (mM) Ki? (mM) Ki/Ki? (63% CI) total of 240 inhibitory curves for combinations of the 12 fluoroquinolones and 5 NSAIDs (at 0, 1, 10 and 100 NFLX 42.9±3.7 12.7 3.39 (0.00041¿27900) PUFX 56.3±4.3 57.4 0.98 (0.689¿1.40) mM), to obtain comprehensive pharmacodynamic CPFX 135±14 27.1 4.98 (0.00014¿173000) parameters. ENX 174±19 17.4 10.0 (0.00012¿245000) AcomparisonofFpred of individual fluoroquinolones GFLX 496±52 257 1.93 (0.0047¿787) in the presence of BPAA allowed us to assess the risk of OFLX 492±54 155 3.18 (0.00048¿21000) convulsion. Briefly, the rank order of F in the TFLX 549±112 509 1.08 (0.346¿3.35) pred LFLX 716±98 152 4.70 (0.00016¿139000) presence of BPAA was PUFXÀENXÀFLRX§NFLXÆ LVFX 1190±193 907 1.31 (0.0712¿24.2) CPFXÀLFLXÆOFLXÆPZFXÀLVFX§GFLXÆSPFX SPFX 1175±188 1326 0.89 (0.200¿3.93) ÆTFLX. Hori et al. have reported doses for convulsion PZFX 2052±486 1790 1.15 (0.207¿6.36) (ED50) after intravenous administration of quinolones FLRX 2106±510 1689 1.25 (0.100¿15.6) concomitantly with 5 nmol BPAA.10) To simply correct Further details as in Table 1.Kdg and g for diclofenac sodium were estimated them by clinical dose, since the ordinary dose in humans as 534±610 and 1.31±6.11, respectively. differs according to the fluoroquinolone, we calculated the ratio of ED50 and the ordinary daily dose of respective 172 Jahye KIM, et al.

Table 6. Predicted unbound drug concentrations of fluoroquinolones and NSAIDs in the human brain after administration of stan- dard dosage

Plasma drug concentrationa Brain distribution d c Cbrain, f Cbrain, f fT100 C b (mg/g brain) (mM) Ref Dose/day ss (max) Ref K (mg/mL) p

CPFX 22 250 mg×3 1.35 28 0.071 (M) 0.170 0.0163 0.0535 ENX 23 200 mg×2 1.97 28 0.077 (M) 0.151 0.0229 0.0715 FLRX 24 200 mg×2 4.32 12 0.300e (R) 0.456 0.591 1.60 GFLX 25 200 mg×2 1.78 37 0.114e (R) 0.107 0.0217 0.0578 LVFX 26 200 mg×3 3.15 16 0.053e (R) 0.140 0.0234 0.0647 LFLX 27 200 mg×3 2.83 19 0.118e (R) 0.111 0.0371 0.105 NFLX 28 200 mg×3 1.05 28 0.057 (M) 0.113 0.00679 0.0213 OFLX 29 200 mg×3 3.33 28 0.054 (M) 0.164 0.0295 0.0816 PZFX 30 500 mg×2 10.9 38 0.040e (R) 0.126 0.0549 0.173 PUFX 31 200 mg×2 1.14 20 0.082e (R) 0.247 0.0231 0.0798 SPFX 32 200 mg×2 1.38 39 0.103e (R) 0.165 0.0235 0.0598 TFLX 33 150 mg×3 0.72 18 0.114e (R) 0.101 0.00829 0.0190 BPAA 6 200 mg×3 4.41 6 0.145 (M) 0.097 0.0620 0.292 Diclofenac 34 25 mg×3 0.28 17 0.055e (R) 0.061 0.000931 0.00315 Loxoprofen 21 60 mg×2 5.00 21 0.044e (R) 0.275 0.0605 0.246 Lornoxicam 35 8 mg×2 0.82 40 0.016e (R) 0.294 0.00386 0.0104 Zaltoprofen 36 80 mg×2 3.60 41 0.019e (R) 0.069 0.00472 0.0158 a Maximum plasma concentration at the steady-state after administration of the standard dosage of each drug (Css (max); mg/mL) was calculated using reference data. b Kp was calculated as the ratio of AUCbrain to AUCplasma in rats (R) or mice (M). c Unbound drug fraction in the whole brain (fT100) was determined by equilibrium dialysis and HPLC, using mouse brain homogenate. d Unbound drug concentration in the human brain (Cbrain, f; mg/g brain) was estimated by applying the equation (7), Cbrain, f＝Css (max)･Kp･fT100. eValues corrected by brain capillary volume of 0.008 mL/g tissue

Table 7. Predicted GABAA receptor occupancy of fluoroquino- BPAA to be ENX, NFLX, and LFLX, CPFXºOFLXº lones in the human brain, after standard dosage administra- SPFX, TFLX. tions of fluoroquinolones with or without NSAIDs Comparison of Fpred of fluoroquinolones alone or with b NSAIDs revealed the potentiation effect of each NSAID Fpred of fluoroquinolones (%) with nothing BPAA zaltoprofen loxoprofen lornoxicam diclofenac on the convulsive activity of the fluoroquinolones. Zal-

PUFX 0.200 7.88 0.200 0.200 0.200 0.200 toprofen, loxoprofen, lornoxicam, and diclofenac sodi- ENX 0.0340 0.734 0.0379 0.0412 0.0340 0.0340 um did not substantially affect Fpred of fluoroquinolones, NFLX 0.0542 0.146 0.0546 0.0548 0.0542 0.0542 suggesting that these four NSAIDs may not enhance the FLRX 0.143 0.143 0.143 0.143 0.143 0.143 risk of convulsion in humans given usual dosage regi- CPFX 0.0510 0.0778 0.0521 0.0555 0.0510 0.0510 mens. Among various NSAIDs, phenylacetic acid and LFLX 0.0393 0.0395 0.0393 0.0393 0.0393 0.0393 OFLX 0.0252 0.0260 0.0252 0.0252 0.0252 0.0252 propionic acid derivatives, such as fenbufen, PZFX 0.0176 0.0177 0.0176 0.0176 0.0176 0.0176 and , have been reported to potently enhance 10) GFLX 0.0101 0.0103 0.0101 0.0102 0.0101 0.0101 the convulsive activity of fluoroquinolones and the inhi- 5) LVFX 0.0108 0.0109 0.0108 0.0108 0.0108 0.0108 bition of GABAA current by fluoroquinolones. However, SPFX 0.00581 0.00597 0.00581 0.00581 0.00581 0.00581 diclofenac, a phenylacetic acid-derived NSAIDs, as well TFLX 0.00453 0.00453 0.00453 0.00453 0.00453 0.00453 as zaltoprofen and loxoprofen, propionic acid-derived a Cbrain, f of fluoroquinolones and NSAIDs are presented in bold type; they are NSAIDs, exhibited far weaker potention than BPAA in mM values converted from mg/g brain values (Table 6) by dividing by the the present study (Fig. 2). These results suggest that each molecular weight of each drug. b Predicted GABAA receptor occupancy of a fluoroquinolone (Fpred)wasgiven NSAID has unique characteristics with regard to drug in- by plugging Cbrain, f and the pharmacodynamic parameters. teraction with quinolones, even among NSAIDs classified in the same group. Oxicam derivates, such as and , have been shown not to act as potentia- 10) drug in humans (ED50/D) and found the rank order of tors of the convulsive activity of fluoroquinolones, in ED50/D to be NFLXºENXÆCPFXºLFLXºGFLXº accordance with the present results for lornoxicam. LVFX. This order is quite consistent with our present However, except for the combination of PUFX and 3) results. Similarly, Akahane et al. reported the rank order BPAA, Fpred were only less than 1%, this being inconsis- of convulsion onset time (min) after intravenous adminis- tent with our previous report in mice showing that block- tration of 25 mg/kg fluoroquinolones with 400 mg/kg ade up to 1–2% or more may be necessary to induce con- Convulsions by Fluoroquinolones and NSAIDs 173

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