Uptake and intracellular activity of an optically active ofloxacin isomer in human neutrophils and tissue culture cells

Vol. 34, No. 2 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 277-280 0066-4804/90/020277-04$02.00/0 Copy igh C 1990, Ame ican Socie y o Mic obiology Up ake and In acellula Ac i i y o an Op ically Ac i e O loxacin Isome in Human Neu ophils and Tissue Cul u e Cells ALVARO PASCUAL,* ISABEL GARCIA, AND EVELIO J. PEREA Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille, Apdo. 914, Se ille41080, Spain Recei ed 28 June 1989/Accep ed 31 Oc obe 1989 The pene a ion o an op ically ac i e o loxacin isome [(-)-o loxacin] in o human neu ophils and di e en issue cul u e cells (HEp-2, McCoy, MDCK, and Ve o) was s udied and compa ed wi h ha o o loxacin by a luo ome ic assay. The cellula - o-ex acellula -concen a ion a ios (CIE) o (-)-o loxacin we e always highe han 6, signi ican ly g ea e han hose o o loxacin a ex acellula concen a ions o 5 and 10 mg/li e . The pene a ion o (-)-o loxacin and o loxacin was doubled when neu ophils we e s imula ed by pho bol my is a e ace a e bu no a ec ed a e inges ion o opsonized S aphylococcus au eus. The CIE a ios o (-)-o loxacin and o loxacin o di e en issue cul u e epi helial cells and ib oblas s we e lowe han hose o neu ophils bu s ill highe han 2. Bo h compounds p oduced a signi ican educ ion in iable in aphagocy ic S. au eus du ing 3 h o exposu e o an imic obial agen s. We conclude ha (-)-o loxacin appea s o each highe in acellula concen a ions han o loxacin, emaining ac i e inside he neu ophils. O loxacin (OFLX) is a quinolone compound ha shows high ac i i y agains a a ie y o g am-posi i e and g am- nega i e bac e ia (17). By using high-pe o mance liquid ch oma og aphy, wo op ically ac i e OFLX isome s ha e been p epa ed: (+)-OFLX and (-)-OFLX (6). The la e isome inhibi ed he supe coiling ac i i y o gy ase om Mic ococcus lu eus 7.4 imes mo e e ec i ely han did he (+)-isome (7). Mo eo e , he in i o ac i i y o (-)-OFLX agains a a ie y o g am-posi i e and g am-nega i e bac e ia was 8 o 128 imes highe han ha o (+)-OFLX and wice as high as ha o acemic OFLX (6). The in acellula pene a ion and ac i i y o an imic obial agen s can be an impo an ac o in he ea men o in ec ions caused by in acellula acul a i e o obliga e pa hogens. Mos s udies on he abili y o an imic obial agen s o pene a e in o cells ha e u ilized phagocy ic cells, and only a ew s udies ha e e alua ed he pene a ion in o o he ypes o cells, such as epi helial cells (1, 2). The e is, howe e , s ong e idence ha some en e oin asi e bac e ia such as en e oin asi e Esche ichia coli su i e in epi helial cells despi e high ex e nal aminoglycoside concen a ions because o he low in acellula pene a ion a e o hese an imic obial agen s (8). We ha e p e iously epo ed ha OFLX pene a es in o human polymo phonuclea leukocy es (PMNs) by an ac i e mechanism and emains ac i e in acellula ly agains S aph- ylococcus au eus (13). Since he an imic obial ac i i y o OFLX seems o be media ed mainly by (-)-OFLX, i would be impo an o s udy whe he his isome can be concen- a ed in o phagocy es and o he euca yo ic cells. The pu pose o his s udy is o e alua e he in acellula pene a ion o his op ically ac i e OFLX isome , (-)- OFLX, in o human PMNs and di e en issue cul u e cells compa ed wi h ha o OFLX. The in acellula ac i i y in PMNs o bo h compounds agains S. au eus is also assessed. MATERIALS AND METHODS Isola ion o PMNs. PMNs we e eco e ed om hepa in- ized enous blood o heal hy dono s and we e pu i ied by p e iously desc ibed me hods (16). PMN p epa a ions we e * Co esponding au ho . 97% pu e. Final cell suspensions we e adjus ed o 5 x 106 PMNs pe ml in Hanks balanced sal solu ion. PMNs we e 95% iable by ypan blue exclusion. Tissue cul u e cells. HEp-2, McCoy, MDCK, and Ve o cells (Flow Labo a o ies, I ine, Uni ed Kingdom) we e g own in minimal essen ial medium (Flow) supplemen ed wi h 1 mM HEPES (N-2-hyd oxye hylpipe azine-N'-2- e hanesul onic acid; Flow) and con aining 10% e al cal se um (Flow) wi hou an ibio ics. Fo each expe imen cells we e de ached om issue cul u e bo les wi h ypsin- EDTA (Flow) and suspended i Hanks balanced sal solu- ion a a concen a ion o 5 x 106 cells pe ml. An imic obial agen up ake by cells. A p e iously de- sc ibed luo ome ic assay o measu e quinolone up ake by human PMNs was used (13). The op ically ac i e o loxacin isome (-)-OFLX was kindly supplied by Daiichi Seiyaku Co., L d. (Tokyo, Japan), and OFLX was supplied by Hoesch (Fede al Republic o Ge many). In hese expe i- men s PMNs o issue cul u e cells we e incuba ed in Hanks balanced sal solu ion con aining di e en concen a ions o he an imic obial agen (2, 5, and 10 mg/li e ). A e di e en incuba ion imes (1.5, 20, and 30 min) a 37°C, cells we e sepa a ed om ex acellula solu ion by cen i uga ion h ough a wa e -impe meable silicone-oil ba ie in a mic o- cen i uge ube (9). The en i e cell pelle , ob ained by cu ing o he po ion o he mic o uge ube con aining he pelle , was placed in 2 ml o 0.1 M glycine-HCl bu e (pH 3.0) and agi a ed igo ously in a o ex shake . Incuba ion o 2 h a oom empe a u e was su icien o elease ully in acellula an imic obial agen . The samples we e cen i uged o 5 min a 5,600 x g, and he amoun o an imic obial agen was de e mined by luo escence emission o he supe na an s wi h a Kon on SFM 25 luo escence spec opho ome e (Kon on, Swi ze land). The luo escence exci a ion and emission maxima o OFLX and (-)-OFLX in 0.1 M glycine- HCl (pH 3.0) we e 292 and 496 nm, espec i ely. Con ols wi hou an imic obial agen s we e always used o de e mine he backg ound luo escence. The in acellula wa e space was measu ed by using i ia ed wa e and he ex acellula ma ke [14C]polye hy- lene glycol (1.4 mCi/g; New England Nuclea Co p., Bos on, Mass.). The cells we e incuba ed wi h hese adiolabeled 277 on Augus 2, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om ANTIMICROB. AGENTS CHEMOTHER. compounds o 2 min a 37°C, and hen cells we e sepa a ed om ex acellula luid by eloci y g adien cen i uga ion as desc ibed abo e and coun ed in a liquid scin illa ion coun e . The o al wa e con en o he cell pelle was co ec ed o apped ex acellula wa e , i.e., polye hylene glycol space, o ob ain he in acellula wa e space. F om he alues ob ained om his p ocedu e, cell-associa ed an imic obial agen concen a ions we e calcula ed and exp essed as a a io o he cellula concen a ion o ex acellula concen a- ion (CIE) (9). In a se ies o expe imen s, OFLX and (-)-OFLX (ex a- cellula concen a ion, 5 mg/li e ) up ake by human PMNs was measu ed a e s imula ion o he cells wi h 200 nM ei he pho bol my is a e ace a e (PMA; Sigma Chemical Co., S . Louis, Mo.) o S. au eus ATCC 25923 opsonized in 5% pooled human se um (15 min, 37°C) a a 10/1 a io o bac e ia o PMNs. PMA o opsonized bac e ia we e added o he PMN suspensions a he same ime as he an imic obial agen s, and he up ake was measu ed as desc ibed abo e. Since he measu emen o OFLX and (-)-OFLX is indi ec and dependen upon luo escence, con ols we e always pe o med o e alua e he e ec o s imuli on he luo es- cence o he an imic obial agen s in cell- ee sys ems. In acelula ac i i y o an imic obial agen s. To e alua e he in acellula ac i i y o an imic obial agen s, a p e iously desc ibed me hod was used (15). B ie ly, 0.1 ml o opsonized bac e ial suspension (5 x 107 CFU/ml) and 0.1 ml o PMNs (5 x 106 PMNs pe ml) we e combined in a se ies o polyp opylene bio ials (Beckman Ins umen s, Inc., Fulle - on, Cali .) and incuba ed in a shake (250 pm) o 60 min a 370C. A e incuba ion, ex acellula bac e ia we e emo ed by di e en ial cen i uga ion. Cells we e hen suspended in 0.2 ml o RPMI medium (GIBCO, Uni ed Kingdom). A his ime (designa ed ime ze o) he di e en an imic obial agen s we e added, and he ials we e incuba ed in a shake (250 pm) a 370C. Vials we e emo ed a ime ze o (con ol) and a e 3 h o incuba ion (con ol and samples wi h an imic o- bial agen s). Cells we e lysed in dis illed wa e , and samples we e dilu ed and pou pla ed in aga . Colonies we e coun ed a e 24 h o incuba ion a 370C. The da a we e exp essed as pe cen ages o su i ing s aphylococci compa ed wi h con- ols (wi hou an imic obial agen s) a ime ze o. In addi ion 10- 9- 8- 7- 0 ~ 6- 4 lx 5 w '-% 4- 3- 2- I- 2 5 10 Ex ocellula concen a ion (mg/I) FIG. 1. OFLX (C1) and (-)-OFLX (O) up ake by human PMNs a di e en ex acellula concen a ions (n = 6). Incuba ions we e done o 20 min. Fo he da a om concen a ions o 5 and 10 mg/li e , P < 0.05. 10 8' w 5 J4 3 2 I 5 10 20 TIME (min) FIG. 2. OFLX (0) and (-)-OFLX (A) up ake by human PMNs a di e en imes (n = 4). The ex acellula concen a ion was 5 mg/li e . o de e mining bac e ial su i al, mo phologic s udies we e also ou inely pe o med a ime ze o and a e 3 h o incuba ion o e alua e he disposi ion o bac e ia (cell asso- cia ed o ex acellula ). Samples o 50 ,ul we e emo ed om bio ials and deposi ed on glass slides. A e s aining wi h W igh s ain, samples we e examined by ligh mic oscopy. All assays we e pe o med in duplica e wi h PMNs om i e di e en dono s. S a is ical analysis o da a. Da a we e exp essed as he means s anda d de ia ions. Di e ences among g oups we e compa ed by analysis o a iance wi h he Bon e oni me hod (18), used o assess s a is ical signi icance a P s 0.05. RESULTS Up ake o an imic obial agen s by PMNS. Figu e 1 shows he CIE a ios o OFLX and (-)-OFLX incuba ed wi h human PMNs a di e en ex acellula concen a ions. Bo h OFLX and (-)-OFLX showed high CIE a ios, being signi - ican ly highe o he la e a ex acellula concen a ions o S and 10 mg/li e . The pene a ion o cell associa ion o OFLX occu ed sligh ly mo e apidly han did ha o (-)-OFLX (Fig. 2). The s imula ion o PMNs by a memb ane ac i a o (PMA) enhanced he in acellula pene a ion o OFLX and (-)- OFLX 2.4 and 1.5 imes, espec i ely (Table 1). The incu- ba ion o PMNs wi h opsonized S. au eus ATCC 25923, howe e , did no modi y he up ake o ei he compound. TABLE 1. E ec o he incuba ion o human PMNs wi h ei he opsonized S. au eus ATCC 25923 o PMA on he up ake o OFLX and (-)-OFLX by hese cells (n = 4)1 CIE a io S imulus OFLX (-)-OFLX None (con ol) 6.0 ± 1.3 8.3 ± 1.7 Opsonized S. au eus 5.2 ± 1.1 7.0 ± 1.6 PMA (200 nM) 14.4 ± 2.3" 13.0 ± 2.3" a Incuba ions we e done o 20 min. The ex acellula concen a ion o OFLX o (-)-OFLX was 5 mg/li e . b p < 0.05 compa ed wi h he con ol. 278 PASCUAL ET AL. on Augus 2, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om OFLOXACIN ISOMER UPTAKE BY HUMAN NEUTROPHILS TABLE 2. OFLX and (-)-OFLX up ake by human PMNs and di e en issue cul u e cells (n =4) An imic o- CIE a io bial agen PMN HEp-2 McCoy MDCK Ve o OFLX 5.8 ± 1.2 2.1 ± 0.2 3.9 ± 1.1 2.1 ± 0.9 3.4 ± 1.2 (-)-OFLX 8.2 1.7b 2.8 ± 0.6 4.7 ± 1.3 1.9 ± 0.9 2.4 ± 0.6 a Incuba ions we e done o 20 min. The ex acellula concen a ion o OFLX o (-)-OFLX was 5 mg/li e . b p < 0.05 compa ed wi h alue o OFLX. Up ake o an imic obial agen s by issue cul u e cells. The CIE a ios o OFLX and (-)-OFLX incuba ed wi h h ee issue cul u e epi helial cells om di e en o igins (HEp-2, McCoy, and MDCK) and one issue cul u e ib oblas cell line (Ve o) o 20 min a 37°C a e shown in Table 2. The CIE a ios o OFLX and (-)-OFLX we e 2 o 3 imes lowe han hose obse ed wi h PMNs, and he e we e no signi ican di e ences be ween he wo compounds. In acellula ac i i ies o an imic obial agen s. The in ac- ellula ac i i ies o di e en concen a ions o OFLX and (-)-OFLX agains S. au eus ATCC 25923 we e e alua ed in a 3-h assay (Table 3). A all o he concen a ions e alua ed, (-)-OFLX p oduced a signi ican educ ion o he su i al o S. au eus in PMNs, simila o ha p oduced by OFLX. DISCUSSION (-)-OFLX is an op ically ac i e OFLX isome ha shows highe in i o ac i i y han (+)-OFLX and he acemic compound OFLX agains di e en mic oo ganisms (6, 7). The pu pose o his s udy was o e alua e whe he his inc eased ac i i y was accompanied by a highe in acellula pene a ion and ac i i y han hose o OFLX. The (-)- OFLX up ake by human PMNs was signi ican ly highe han ha o OFLX a ex acellula concen a ions o 5 and 10 mg/li e . This di e ence was no obse ed a a lowe con- cen a ion (2 mg/li e ). Ne e heless, bo h compounds eached high in acellula concen a ions in hese phago- cy es, as has been desc ibed o o he quinolones wi h di e en me hodology (3, 10). The pene a ion o (-)-OFLX and OFLX was no a ec ed when PMNs we e incuba ed wi h opsonized S. au eus. This has been desc ibed o o he an imic obial agen s such as e y h omycin, i ampin, and gen amicin (5). Howe e , he TABLE 3. E ec o OFLX and (-)-OFLX on he su i al o S. au eus ATCC 25923 wi hin human PMNs (n = 5) An imic obial agen and % Su i al o concn (mg/li e ) S. au eus a 3 h No agen 45.5 ± 5.2 OFLX 0.5 20.9 ± 3. la 2 17.6 ± 3.0O 5 15.0 ± 2.7a 10 5.8 ± 1.4a (-)-OFLX 0.5 17.8 ± 3.2a 2 15.5 ± 3. la 5 12.2 ± 2.6a 10 9.1 ± 2.1la a P < 0.05 compa ed wi h he con ol. pene a ion o o he an imic obial agen s, such as clindamy- cin, has been shown o inc ease a e phagocy osis o S. au eus (5). The s imula ion o PMN memb anes by PMA, howe e , doubled he up ake o bo h compounds by human PMNs, poin ing ou ha he pene a ion o (-)-OFLX in o PMNs could be media ed by an ac i e mechanism, as has been demons a ed o o loxacin (13), since PMA ac i a es he NADPH oxidase sys em ia p o ein kinase C (11). To s udy he pene a ion o hese an imic obial agen s in o o he euca yo ic cells, we used a issue cul u e cell model. The pene a ion o bo h compounds in o issue cul u e epi helial cells and ib oblas s was signi ican ly lowe han ha ob- se ed in PMNs bu s ill eached in acellula concen a ions ha we e a leas double he ex acellula concen a ions. The use o his model o e s an in e es ing insigh in o he pene a ion o an ibac e ial subs ances in o cells and may ha e ele ance o human in ec ions p oduced by mic oo - ganisms ha . a e able o su i e and e en mul iply wi hin epi helial cells. Con as s be ween phagocy e an imic obial agen up ake and in acellula ac i i y ha e been desc ibed o a ew an imic obial agen s (5, 15). In ou s udy, (-)-OFLX showed a good in acellula pene a ion in o and ac i i y in PMNs agains S. au eus. The in acellula an imic obial ac i i y o his isome was simila o ha obse ed o he acemic compound and simila o hose desc ibed o o he quino- lones agains di e en bac e ia (4,12, 14). In summa y, (-)-OFLX, an op ically ac i e OFLX iso- me , pene a ed in o human PMN, eaching concen a ions highe han - hose eached by OFLX and emaining ac i e in acellula ly. This compound also showed a high pene a- ion in o issue cul u e epi helial cells and ib oblas s. The high an imic obial ac i i y o his compound, highe e en han ha o he acemic compound OFLX, added o he p ope ies obse ed in his s udy, could jus i y addi ional s udies o e alua e he possible clinical ele ance o hese indings. ACKNOWLEDGMENTS We hank M. C. Guzman o echnical assis ance and C. Acos a o yping he manusc ip . LITERATURE CITED 1. B own, K. N., and A. Pe ci al. 1978. Pene a ion o an imic o- bials in o issue cul u e cells and leukocy es. Scand. J. In ec . Dis. 14(Suppl.):251-260. 2. Chadwick, P. R., and A. R. Meile sh. 1987. The use o a issue cul u e model o assess he pene a ion o an ibio ics in o epi helial cells. J. An imic ob. Chemo he . 19:211-220. 3. Easmon, C. S. F., and J. P. C ane. 1985. Up ake o cip o loxacin by mac ophages. J. Clin. Pa hol. 38:442-444. 4. Easmon, C. S. F., J. P. C ane, and A. Blowe s. 1986. E ec o cip o loxacin on in acellula o ganisms: in- i o and in- i o s udies. J. An imic ob. Chemo he . 18(Suppl. D):43-48. 5. Hand, W. L., and N. L. King-Thompson. 1986. Con as be- ween phagocy e an ibio ic up ake and subsequen in acellula bac e icidal ac i i y. An imic ob. Agen s Chemo he . 29:135- 140. 6. Hayakawa, I., S. A a ashi, S. Yokohama, M. Imamu a, K. Sakano, and M. Fu ukawa. 1986. Syn hesis and an ibac e ial ac i i ies o op ically ac i e o loxacin. An imic ob. Agen s Chemo he . 29:163-164. 7. Imamu a, M., S. Shibamu a, I. Hayakawa, and Y. Osada. 1987. Inhibi ion o DNA gy ase by op ically ac i e o loxacin. An imi- c ob. Agen s Chemo he . 31:325-327. 8. Kihis om, E., and L. Andake . 1985. Inabili y o gen amicin and VOL. 34, 1990 279 on Augus 2, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om ANTIMICROB. AGENTS CHEMOTHER. os omycin o elimina e in acellula En e obac e iaceae. J. An imic ob. Chemo he . 15:723-728. 9. Klempne , M. S., and B. S y . 1981. Clindamycin up ake by human neu ophils. J. In ec . Dis. 144:472475. 10. Koga, H. 1987. High-pe o mance liquid ch oma og aphy mea- su emen o an imic obial concen a ions in polymo phonuclea leukocy es. An imic ob. Agen s Chemo he . 31:1904-1908. 11. Ma idonneau-Pa ini, I., S. M. T ingale, and A. I. Taube . 1986. Iden i ica ion o dis inc ac i a ion pa hways o he human neu ophil NADPH-oxidase. J. Immunol. 137:2925-2929. 12. Mila o ic, D. 1986. In aphagocy ic ac i i y o cip o loxacin and CI 934. Eu . J. Clin. Mic obiol. 5:659-660. 13. Pascual, A., I. Ga cia, and E. J. Pe ea. 1989. Fluo ome ic measu emen o o loxacin up ake by human polymo phonuclea leukocy es. An imic ob. Agen s Chemo he . 33:653-656. 14. Pascual, A., L. Ma inez-Ma inez, and E. J. Pe ea. 1989. E ec o cip o loxacin and o loxacin on human polymo phonuclea leukocy e ac i i y agains s aphylococci. Chemo he apy (Basel) 35:17-22. 15. Pascual, A., D. Tsukayama, J. Ko a ik, G. Gekke , and P. K. Pe e son. 1987. Up ake and ac i i y o i apen ine in human pe i oneal mac ophages and polymo phonuclea leukocy es. Eu . J. Clin. Mic obiol. 6:152-157. 16. Pe e son, P. K., J. Ve hoe , D. Schmeling, and P. G. Quie. 1977. Kine ics o phagocy osis and bac e ial killing by human poly- mo phonuclea leukocy es and monocy es. J. In ec . Dis. 136: 502-509. 17. Salo, K., Y. Ma suu a, M. Ino e, T. Une, Y. Osada, H. Ogawa, and S. Mi suhashi. 1982. In i o and in i o ac i i y o DL-8280, a new oxazine de i a i e. An imic ob. Agen s Che- mo he . 22:548-553. 18. Wallens ein, S., C. L. Zucne , and J. L. Feiss. 1980. Some s a is ical me hods use ul in ci cula ion esea ch. Ci c. Res. 47:1-9. 280 PASCUAL ET AL. on Augus 2, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om