Uptake and intracellular activity of trovafloxacin in human phagocytes and tissue-cultured epithelial cells

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 0066-4804/97/$04.0010Feb. 1997, p. 274–277 Vol. 41, No. 2 Copy igh q1997, Ame ican Socie y o Mic obiology Up ake and In acellula Ac i i y o T o a loxacin in Human Phagocy es and Tissue-Cul u ed Epi helial Cells ALVARO PASCUAL,* ISABEL GARCI ´A, SOFI ´A BALLESTA, AND EVELIO J. PEREA Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille, Se ille 41080, Spain Recei ed 28 May 1996/Re u ned o modi ica ion 9 Sep embe 1996/Accep ed 12 No embe 1996 The pene a ion o o a loxacin in o human polymo phonuclea leukocy es (PMNs), human pe i oneal mac ophages, and issue-cul u ed epi helial cells (McCoy cells) was e alua ed. The cellula concen a ion o ex acellula concen a ion (C/E) a ios o o a loxacin we e g ea e han 9 o ex acellula concen a ions anging om 0.5 o 25 mg/ml. The up ake o o a loxacin by PMNs was apid, e e sible, nonsa u able, no ene gy dependen , and signi ican ly inc eased a pH 6. The C/E a ios o o a loxacin we e no a ec ed by cell iabili y bu we e signi ican ly inc eased a 4&C. Inges ion o opsonized zymosan, bu no opsonized S aphylo- coccus au eus, signi ican ly inc eased he amoun o PMN-associa ed o a loxacin. This agen a concen a- ions o 0.5 and 1 mg/ml induced a g ea e educ ion in he su i al o in acellula S. au eus in PMNs han cip o loxacin and o loxacin. I was concluded ha o a loxacin eaches concen a ions wi hin phagocy ic and nonphagocy ic cells se e al imes highe han he ex acellula ones, while i emains ac i e in PMNs. Mos luo oquinolones a e able o concen a e in phagocy ic and nonphagocy ic cells, eaching concen a ions se e al imes highe han he ex acellula ones (9). Mo eo e , hese agen s ha e been shown o emain ac i e in acellula ly agains di - e en acul a i e in acellula pa hogens. Mos s udies on he in acellula pha macology o an imi- c obial agen s ha e used human phagocy es, and only a ew s udies ha e e alua ed he pene a ion in o o he ypes o cells. Ne e heless, he pa hogenic mechanism o se e al in ec ions in ol es he su i al o bac e ia in nonphagocy ic cells, as has been desc ibed o Chlamydia achoma is and en e oin asi e Esche ichia coli in epi helial cells. An impo an limi a ion o mos comme cially a ailable luo oquinolones is hei limi ed ac i i y agains g am-posi i e pa hogens. In ecen yea s, howe e , new quinolones ha o e good ac i i y agains hese bac e ia ha e been desc ibed. Among hem, spa loxacin has been shown o pene a e apidly in o phagocy ic cells, emaining ac i e agains S aphylococcus au eus (4). T o a loxacin (CP-99,219) is a new azabicyclo naph hy i- done agen wi h a b oad spec um o an ibac e ial ac i i y which includes g am-posi i e bac e ia such as S ep ococcus pneumoniae and S. au eus (6). Mo eo e , his agen showed high in i o ac i i y agains Chlamydia achoma is (2). The pu pose o his s udy was o e alua e he up ake o o a loxacin by human polymo phonuclea leukocy es (PMNs), human pe i oneal mac ophages (PM s), and issue-cul u ed epi helial cells. The mechanism in ol ed in he pene a ion o his agen in o human PMNs and i s in acellula ac i i y com- pa ed wi h hose o cip o loxacin and o loxacin we e also e al- ua ed. MATERIALS AND METHODS Isola ion o phagocy es. PMNs we e eco e ed om he hepa inized enous blood o heal hy dono s and we e pu i ied by p e iously desc ibed me hods (10). PMN p epa a ions we e .97% pu e. PM s we e isola ed om pe i oneal e lu- en s o pa ien s unde going con inuous ambula o y pe i oneal dialysis (CAPD) and who we e being ollowed up by he Neph ology Depa men o he Uni e - si y Hospi al o Se ille, as desc ibed p e iously (13). Dono s we e clinically unin ec ed a he ime o he s udy. Cell p epa a ions om he CAPD dono always con ained .75% PM s and ,15% PMNs. Final cell suspensions we e adjus ed o 2 310 6 PM s/ml o 2 310 6 PMNs/ml in Hanks balanced sal solu ion (HBSS) con aining 0.1% gela in. Bo h popula ions o cells we e $95% iable by ypan blue exclusion c i e ia. Tissue cul u e cells. McCoy cells (Flow Labo a o ies, I ine, Uni ed King- dom) we e g own in minimal essen ial medium (Flow) supplemen ed wi h 1 mM HEPES (N-2-hyd oxye hylpipe azine-N9-2-e hanesul onic acid; Flow) and con- aining 10% e al cal se um (Flow) wi hou an ibio ics. Fo each expe imen , he cells we e de ached om he issue cul u e bo les wi h ypsin-EDTA (Flow), washed once wi h minimal essen ial medium con aining e al cal se um (10%), and suspended in HBSS a a concen a ion o 5 310 6 cells pe ml. T o a loxacin up ake by cells. Up ake o adiolabeled o a loxacin by phago- cy ic and epi helial cells was de e mined by a eloci y g adien cen i uga ion echnique desc ibed by Klempne and S y (7). [ 14 C] o a loxacin (21.53 mCi/ mg) and o a loxacin we e kindly supplied by P ize Cen al Resea ch (G o on, Conn.). In hese expe imen s, phagocy es o issue cells we e incuba ed in HBSS con aining di e en concen a ions o o a loxacin (0.5, 1, 2, 5, 10, and 25 mg/ml). A e di e en incuba ion pe iods a 378C, he cells we e sepa a ed om he ex acellula solu ion by cen i uga ion h ough a wa e -impe meable silicone oil ba ie in a mic ocen i uge ube. A 10-ml aliquo o he ex acellula medium and he en i e cell pelle , ob ained by cu ing o he po ion o he mic ocen i- uge ube con aining he pelle , we e placed in 3 ml o scin illa ion luid (Ready Mic o; Beckman Ins umen s, Inc., Fulle on, Cali .), and he adioac i i y was coun ed in a liquid scin illa ion coun e (model LS 1801; Beckman). A e de- e mina ion o he cell olume wi h adiolabeled polye hylene glycol and wa e (New England Nuclea ), he a e o accumula ion o he an imic obial agen in PMNs, PM s, o issue cells was calcula ed and exp essed as cellula concen- a ion o ex acellula concen a ion (C/E) a io (13). Cha ac e iza ion o o a loxacin up ake. Fu he s udies o elucida e he mechanism o o a loxacin up ake by PMNs we e pe o med as desc ibed p e- iously (10). The impo ance o cell iabili y was s udied by using PMNs killed by exposu e o 10% o malin o 30 min. These cells we e hen washed and sus- pended in esh medium. Mo eo e , he in luence o en i onmen al empe a u e, pH, and me abolic inhibi o s was e alua ed. The in luence o empe a u e was examined by compa ing an imic obial up ake a 4 and 378C. The pH p o iles o o a loxacin up ake in media p eadjus ed o di e en ex e nal pHs (pH 6, 7, and 8) by he addi ion o 10 N HCl o 10 N NaOH we e measu ed. An inhibi o o glycolysis (sodium luo ide, 1.5 310 23 M; Sigma Chemical Co., S . Louis, Mo.), an inhibi o o mi ochond ial oxida i e me abolism (sodium cyanide; 1.5 310 23 M; Sigma), a blocke o he p o on g adien (ca bonyl cyanide m-chlo ophenyl- hyd azone; 1.5 310 25 M; Sigma), and an uncouple o oxida i e phospho yla- ion (2,4-dini ophenol; 1 310 24 M; Sigma) we e used as me abolic inhibi o s. PMNs in HBSS wi h and wi hou me abolic inhibi o s we e incuba ed o 30 min a 378C, o a loxacin ( inal concen a ion, 2 mg/ml) was hen added, and he up ake was measu ed, as desc ibed abo e. In a se ies o expe imen s, o a loxacin (ex acellula concen a ion, 2 mg/ml) up ake by human PMNs was measu ed a e he s imula ion o cells wi h 200 nM pho bol my is a e ace a e (PMA; Sigma) and a e he phagocy osis o ei he opsonized zymosan (0.9 mg/li e ; Sigma) o S. au eus ATCC 25923, opsonized in 5% pooled human se um (15 min a 378C) a a 10/1 a io o bac e ia o PMNs. * Co esponding au ho . Mailing add ess: Depa men o Mic obi- ology, School o Medicine, Uni e si y o Se ille, Apdo. 914, Se ille 41080, Spain. Phone: 34.5.4557448. Fax: 34.5.4377413. 274 on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om PMA o opsonized pa icles we e added o PMN suspensions a he same ime ha he an imic obial agen was added, and he up ake was measu ed as de- sc ibed abo e. The e lux o e e sibili y o he binding o cell-associa ed o a loxacin was also s udied. PMNs, PM s, o McCoy cells we e incuba ed o 10 min a 378C wi h o a loxacin (ex acellula concen a ion, 2 mg/ml), collec ed by cen i u- ga ion, and apidly suspended in quinolone- ee medium. Cell-associa ed o a- loxacin was quan i ied a a ious ime in e als (5, 10, and 20 min) a e he emo al o he ex acellula an imic obial agen . All assays we e pe o med in duplica e wi h PMNs om i e di e en dono s. Pa i ion coe icien o quinolones. The pa i ion coe icien s o quinolones we e de e mined by he modi ied me hod o Nikaido (8). Solu ions (10 mg/ml) o quinolones we e made in 0.1 M phospha e bu e (pH 7.2). A e being shaken wi h an equal olume o n-oc anol a 258C o 48 h and cen i uged a 1,870 3g o phase sepa a ion, he concen a ions o quinolones in he aqueous phase we e de e mined by a spec opho ome ic assay measu ing he A 260 o o a- loxacin and he A 286 o o loxacin (Hoechs AG, Ba celona, Spain). The pa i- ion coe icien s we e exp essed as he a io o he amoun o he compound in he n-oc anol o ha in he aqueous phase. O ganisms and suscep ibili y es ing. S. au eus ATCC 25923 was used o he killing assays. Suscep ibili y s udies we e de e mined by dilu ion assay. The MICs and minimum bac e icidal (MBCs) o cip o loxacin (Baye AG, Le e kusen, Ge many), o loxacin (Hoechs AG), concen a ions and o a loxacin o his s ain we e 0.25, 0.25, and 0.03 mg/ml, espec i ely. In acellula ac i i ies o an imic obial agen s. To e alua e he in acellula ac i i ies o an imic obial agen s, a p e iously desc ibed me hod was used (13). B ie ly, 0.1 ml o opsonized bac e ial suspension (5 310 7 CFU/ml) and 0.1 ml o PMNs (5 310 6 PMNs pe ml) we e combined in a se ies o polyp opylene bio ials (Beckman), and he ials we e incuba ed in a shake (250 pm) o 60 min a 378C. A e incuba ion, he mix u es we e washed h ee imes wi h 2.5 ml o ice-cold phospha e-bu e ed saline by using di e en ial cen i uga ion (160 3 g; 5 min a 48C) o emo e he ex acellula bac e ia. The cells we e hen suspended in 0.2 ml o RPMI medium (Sigma). A his ime, di e en concen- a ions o he di e en luo oquinolones we e added, and he ials we e incu- ba ed in a shake (50 pm) a 378C. The ials we e emo ed a ime ze o and a e 3 h o incuba ion (con ol and samples wi h an imic obial 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 378C. The da a we e exp essed as he pe cen ages o s aphylococci su i ing compa ed wi h he le els in he con ols (wi hou an imic obial agen s) a 3 h. In addi ion 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 3ho incuba ion o e alua e he disposi ion o he bac e ia (cell associa ed o ex acellula ). Samples (50 ml) we e emo ed om he bio ials and we e deposi ed on glass slides. A e being s ained wi h W igh s ain, he 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. The da a we e exp essed as he means 6s anda d de ia ions. Di e ences among g oups we e compa ed by analysis o a iance, used o assess s a is ical signi icance a P#0.05. RESULTS Up ake o o a loxacin by PMNs, PM s, and McCoy cells. Figu e 1 shows he kine ics o up ake o o a loxacin by hu- man PMNs. T o a loxacin up ake by hese cells was apid and high. Wi h ex acellula concen a ions o 2 mg/ml, he C/E a ios we e g ea e han 10 a e 1 min o incuba ion. The e ec o he ex acellula concen a ions o o a loxacin on up ake by PMNs is p esen ed in Fig. 2. Cell-associa ed o a loxacin was no sa u able a ex acellula concen a ions anging om 0.5 o 25 mg/ml. The up ake o o a loxacin by human PM s and issue-cul u ed epi helial cells was simila o ha ob ained wi h PMNs. A ex acellula concen a ions o 2 mg/ml (30 min; 378C), he C/E a ios we e 10.6 62.0 o PMNs, 10.2 62.0 o PM s, and 9.6 61.9 o McCoy cells. The e ec s o pH, en i onmen al empe a u e, cell iabili y, and me abolic inhibi o s on he up ake o o a loxacin by human PMNs a e indica ed in Table 1. T o a loxacin up ake by PMNs was signi ican ly highe a pH 6 and when iable PMNs a 48C we e used. None o he me abolic inhibi o s used a ec ed he in acellula pene a ion o his quinolone. The s imula ion o PMNs by a memb ane ac i a o (PMA) and he phagocy osis o opsonized S. au eus cells did no a ec he in acellula pene a ion o o a loxacin (C/E a ios, 9.0 6 1.7 and 8.2 61.6, espec i ely). The phagocy osis o opsonized zymosan, howe e , signi ican ly inc eased he up ake o o a- loxacin by PMNs (C/E a io, 13.9 61.7; P,0.05). To e alua e whe he o a loxacin ha had been aken up by human PMNs, PM s, and issue-cul u ed epi helial cells was igh ly bound o cellula componen s, we e alua ed he kine ics o e lux (Fig. 3). The e e sibili y o binding o o a loxacin was apid, wi h 80, 65, and 72% o he cell-associa ed d ug being los by 5 min in PMNs, PM s, and McCoy cells, espec- i ely. The kine ics o he e lux o o a loxacin om PMNs a 378C was simila o ha obse ed in PMNs a 48C o dead PMNs a 378C (da a no shown). Pa i ion coe icien o quinolones. The pa i ion coe icien s o o a loxacin and o loxacin we e 0.646 and 0.168, espec- i ely. This means ha o a loxacin is app oxima ely ou imes mo e hyd ophobic han o loxacin. In acellula ac i i y o o a loxacin. The in acellula ac- i i y o o a loxacin agains S. au eus compa ed wi h hose o cip o loxacin and o loxacin was e alua ed by a 3-h assay (Fig. 4). A ex acellula concen a ions o 0.5 and 1 mg/ml, he in acellula ac i i y o o a loxacin was signi ican ly g ea e han hose o cip o loxacin and o loxacin. FIG. 1. Kine ics o o a loxacin up ake by human PMNs (n55). The ex acellula concen a ion was 2 mg/ml. E o ba s indica e s anda d de ia ions. FIG. 2. T o a loxacin up ake by human PMNs a di e en ex acellula con- cen a ions (n55). Incuba ions we e ca ied ou o 20 min. E o ba s indica e s anda d de ia ions. VOL. 41, 1997 UPTAKE OF TROVAFLOXACIN BY PHAGOCYTES 275 on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om DISCUSSION The up ake o adiolabeled o a loxacin by phagocy ic cells (PMNs and PM s) and nonphagocy ic cells has been e alu- a ed. A he apeu ic ex acellula concen a ions, o a loxacin eached concen a ions in PMNs 10 o mo e imes highe han he ex acellula concen a ions. These alues we e sligh ly highe han hose obse ed wi h o he luo oquinolones such as o loxacin, lome loxacin, spa loxacin, and BAY-Y-3118 (4, 5, 11, 12). This could be ela ed o he ac ha o a loxacin is mo e hyd ophobic han hose quinolones and heo e ically could pass mo e easily h ough he cell memb ane. The up ake o o a loxacin by PM s was simila o ha obse ed o PMNs. This inding could be in e es ing since in aleukocy ic su i al o bac e ia has been pos ula ed as an impo an cause o pe sis en pe i oni is in pa ien s unde going CAPD (1) and li le in o ma ion on he pene a ion o an imi- c obial agen s in human PM s is a ailable. A highe ex acel- lula concen a ions (10 mg/ml) and unde di e en expe imen- al condi ions, Edels ein e al. (3) epo ed C/E a ios o o a loxacin o g ea e han 20 o guinea pig al eola mac- ophages. T o a loxacin also eached high in acellula concen a ions (C/E a ios, $9) in issue-cul u ed epi helial cells. These alues a e highe han hose obse ed o o loxacin and spa loxacin (4, 11). Since o a loxacin yielded high in insic ac i i y agains C. achoma is and since hese mic oo ganisms mul iply wi hin epi helial cells, hese esul s could ein o ce he po en- ial use o his agen agains chlamydial in ec ions. The up ake o o a loxacin by human PMNs was apid, nonsa u able (ex acellula concen a ions anged om 0.5 o 25 mg/ml), e e sible (as was he case o PM s and McCoy cells), and no ene gy dependen . I was signi ican ly inc eased a acidic pH, as was epo ed elsewhe e o o he quinolones (4, 5), bu o a lesse ex en . This phenomenon could be ela ed o he ac ha such quinolones display a ee ca boxyl g oup ha becomes p o ona ed when he pH changes om alkaline o acidic. I has been obse ed ha he ionized o ms o weak o ganic acids di use h ough biological memb anes much mo e slowly han hei un-ionized o ms (14). The up ake o o a loxacin by human PMNs was signi ican ly inc eased a 48C and was no a ec ed by cell iabili y. We ha e p e iously obse ed ha he up ake o some quinolones such as ema- loxacin and lome loxacin signi ican ly dec eased a 48C and was a ec ed by cell iabili y (12), while he up ake o o he quinolones such as spa loxacin was no a ec ed by hese pa- ame e s (4). We ha e no p e iously obse ed such a ema k- able inc ease a 48C wi h o he quinolones. These da a could indica e ha mo e han one mechanism may media e he in- acellula pene a ion o di e en quinolones. Fo o a loxa- cin, mos da a show ha a passi e mechanism, such as ha desc ibed o spa loxacin (4), is in ol ed. The e ec s o phagocy osis and s imula ion o he cell mem- b ane on he up ake o o a loxacin by human PMNs was simila o hose p e iously obse ed wi h spa loxacin (4). The inges ion o opsonized S. au eus cells did no a ec he in a- cellula pene a ion o o a loxacin, bu he inges ion o op- sonized zymosan signi ican ly inc eased he C/E a io o o a- loxacin. Ne e heless, he up ake o o a loxacin was s ill high unde bo h condi ions. A ex acellula concen a ions o 0.5 and 1 mg/ml, o a- loxacin showed ac i i y agains S. au eus g ea e han hose o cip o loxacin and o loxacin in human PMNs. This e ec was pa ially due o a highe in insic ac i i y o o a loxacin agains he s ain ha we used. In summa y, o a loxacin pene a es in o phagocy ic and nonphagocy ic cells, eaching in acellula concen a ions se - e al imes g ea e han he ex acellula ones, while i emains FIG. 3. E lux o o a loxacin om human PMNs, PM s, and McCoy cells (n53). A e incuba ion wi h 2 mg o o a loxacin pe ml o 20 min, he cells we e washed and esuspended in an imic obial agen - ee medium. Cell-associ- a ed o a loxacin was hen measu ed a di e en imes. FIG. 4. Ac i i y o o a loxacin compa ed o hose o cip o loxacin and o loxacin agains in acellula S. au eus in human PMNs (n55). Da a a e exp essed as pe cen ages o su i ing bac e ia compa ed o ha o he con ol. E o ba s indica e s anda d de ia ions. *, P, 0.05 compa ed o cip o loxacin and o loxacin. TABLE 1. E ec s o pH, cell iabili y, en i onmen al empe a u e, and me abolic inhibi o s on he in acellula pene a ion o o a loxacin in human PMNs Condi ion a C/E a io pH 7.2 (con ol)......................................................................11.3 60.8 pH 6 .........................................................................................16.5 60.4 b pH 8 .........................................................................................10.3 61.0 Viable cells, 48C......................................................................62.0 614.0 b Dead cells, 378C......................................................................14.4 64.3 Sodium luo ide ......................................................................11.1 61.7 Sodium cyanide....................................................................... 9.3 61.8 Ca bonyl cyanide m-chlo ophenyld azone.......................... 9.7 61.7 2,4-Dini ophenol ...................................................................13.4 63.5 a Expe imen s we e ca ied ou o 20 min a an ex acellula concen a ion o 2mg/ml wi h PMNs om i e subjec s. b P,0.05 compa ed wi h he con ol. 276 PASCUAL ET AL. 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