Intracellular penetration and activity of BAY Y 3118 in human polymorphonuclear leukocytes

ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, OC . 1994, p. 2426-2429 0066-4804/94/$04.00+0 Copy igh ©) 1994, Ame ican Socie y o Mic obiology In acellula Pene a ion and Ac i i y o BAY Y 3118 in Human Polymo phonuclea Leukocy es ISABEL GARCIA, ALVARO PASCUAL,* AND EVELIO J. PEREA Depa men o Mic obiology, Uni e si y o Se ille School o Medicine, 41080-Se ille, Spain Recei ed 28 Oc obe 1993/Re u ned o modi ica ion 10 May 1994/Accep ed 19 July 1994 The pene a ion o a new quinolone (BAY Y 3118) in o human polymo phonuclea leukocy es (PMNs) was e alua ed by a luo ome ic assay. The cellula concen a ion- o-ex acellula concen a ion (C/E) a io was highe han 6.3 a ex acellula concen a ions anging om 2 o 100 mg/li e . The up ake o BAY Y 3118 was apid, e e sible and nonsa u able. The in acellula pene a ion o BAY Y 3118 was signi ican ly a ec ed by en i onmen al empe a u e (C/E a io a 4°C, 5.4 ± 0.5; con ol, 7.5 ± 0.9; P < 0.05) and cell iabili y (C/E a io in dead PMNs, 5.5 ± 0.8; con ol, 7.5 ± 0.9; P < 0.05), bu i was no a ec ed by me abolic inhibi o s. The inges ion o opsonized zymosan o opsonized S aphylococcus au eus signi ican ly dec eased he le els o PMN-associa ed BAY Y 3118. Cell s imula ion by a memb ane ac i a o , howe e , signi ican ly inc eased he in acellula concen a ion o his quinolone. A he apeu ic ex acellula concen a ions (0.5, 2, and 5 mg/li e ), BAY Y 3118 showed in acellula ac i i y g ea e han ha o cip o loxacin agains S. au eus in human PMNs. I was concluded ha BAY Y 3118 eaches high in acellula concen a ions wi hin human PMNs and emains ac i e in acellula ly. The en y o an imic obial agen s in o phagocy ic cells is essen ial o ac i i y agains in acellula mic oo ganisms. The use o an imic obial agen s ha each high in acellula con- cen a ions and emain ac i e wi hin phagocy es could be impo an in he he apy o in ec ions caused by acul a i e in acellula bac e ia (2). Mos o he new quinolones a e able o pene a e and each high in acellula concen a ions in di e en phagocy ic cells. Among hem, no loxacin, cip o loxacin, o loxacin, and le o- loxacin ha e been shown o be e ec i e agains ypical acul- a i e in acellula pa hogens, such as mycobac e ia o Legio- nella spp., and agains o he bac e ia, such as S aphylococcus au eus, ha in ce ain ci cums ances a e able o su i e wi hin phagocy ic cells (4, 5, 10). BAY Y 3118 is a new chlo o luo oquinolone wi h an ibac- e ial ac i i y agains an expanded spec um, including g am- posi i e cocci and anae obic bac e ia (14). I was ound o be mo e ac i e han cip o loxacin agains S. au eus, including cip o loxacin- esis an and me hicillin- esis an species (1). The pu pose o his s udy was o e alua e he up ake o BAY Y 3118 by human polymo phonuclea leukocy es (PMNs). The mechanism in ol ed in he pene a ion o hese agen s in o human PMNs and i s in acellula ac i i y agains S. au eus compa ed wi h ha o cip o loxacin we e also e alua ed. MATERIALS AND METHODS Isola ion o PMNs. PMNs we e eco e ed om hepa inized enous blood o heal hy dono s and pu i ied by p e iously desc ibed me hods (13). PMN p epa a ions we e 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. BAY Y 3118 up ake by PMNs. A p e iously desc ibed luo ome ic assay was used o measu e quinolone up ake by human PMNs (9). BAY Y 3118 and cip o loxacin we e kindly * Co esponding au ho . Mailing add ess: Depa men o Mic obi- ology, Uni e si y o Se ille School o Medicine, Apdo. 914, 41080- Se ille, Spain. Phone: 34.5.4557448. Fax: 34.5.4377413. supplied by Baye A.G., Le e kusen, Ge many. In hese ex- pe imen s, PMNs we e incuba ed in Hanks balanced sal solu ion con aining di e en concen a ions o an imic obial agen (2 o 100 mg/li e ). A e di e en incuba ion imes 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 ocen i uge ube. The en i e cell pelle , ob ained by cu ing o he po ion o he mic ocen i uge ube con ain- ing 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 he in acellula an imic obial agen ully (9). Samples we e cen- i uged o 5 min a 5,600 x g, and he amoun o an imic o- bial agen was de e mined by luo escence emission o supe - na an s wi h an F 2000 luo escence spec opho ome e (Hi achi, Tokyo, Japan). The luo escence exci a ion and emis- sion maxima in 0.1 M glycine-HCl (pH 3.0) we e 285 and 459 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. In acellula wa e space was measu ed by using ia ed wa e and he ex acellula ma ke [14C]polye hylene glycol (1.4 mCi/g; New England Nuclea Co p., Bos on, Mass.). Cells we e incuba ed wi h hese adiolabeled compounds o 2 min a 37°C, 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 . To 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 by his p ocedu e, cell-associa ed an imic o- bial agen concen a ions we e calcula ed and exp essed as a ios o he cellula concen a ion o ex acellula concen a- ion (C/E a ios) (7). Cha ac e iza ion o BAY Y 3118 up ake. Fu he s udies o elucida e he mechanism o BAY Y 3118 up ake o PMNs we e pe o med as desc ibed p e iously (9). The impo ance o cell iabili y was s udied by using PMNs killed by exposu e o 10% Fo malin o 30 min. These cells we e washed and hen suspended in esh medium. Mo eo e , he in luences o en i onmen al empe a u e, pH, and me abolic inhibi o s we e e alua ed. The in luencc o empe a u e was examined by 2426 Vol. 38, No. 10 on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om BAY Y 3118 UPTAKE BY NEUTROPHILS 2427 20 c) E 15 c .I.. c 0 co ,1 5 0 co L6 0W 0 5 10 20 25 30 40 (min) FIG. 1. BAY Y 3118 up ake by human PMNs and e lux o PMN-associa ed BAY Y 31118 a e he emo al o he ex acellula d ug (n = 5). The ex acellula concen a ion was 2 mg/li e . E o ba s indica e s anda d de ia ions. compa ing an imic obial up ake a 4 and 37°C. The pH p o iles o BAY Y 3118 up ake in media p eadjus ed o di e en ex e nal pHs (pH 5, 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 x 10-3 M) (Sigma Chemical Co., S . Louis, Mo.), an inhibi o o mi ochond ial oxida i e me abolism (sodium cyanide, 1.5 x 10-3 M) (Sigma), a blocke o he p o on g adien (ca bonyl cyanide m-chlo ophenylhyd azone, 1.5 x 10-' M) (Sigma), and an uncouple o oxida i e phospho yla- ion (2,4-dini ophenol, 1 x 10-' M) (Sigma) we e used as me abolic inhibi o s. PMNs in Hanks balanced sal solu ion wi h and wi hou me abolic inhibi o s we e incuba ed o 30 min a 37°C. BAY Y 3118 ( inal concen a ion, 2 mg/li e ) was hen added, and he up ake was measu ed as desc ibed abo e. In a se ies o expe imen s, BAY Y 3118 (ex acellula concen a ion, 2 mg/li e ) up ake by human PMNs was mea- su 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, 37°C) a a 10/1 a io o bac e ia o PMNs. PMA o opsonized pa icles we e added o PMN suspensions a he same ime as he an imic obial agen , and he up ake was measu ed as desc ibed abo e. The e iux o e e sibili y o he binding o PMN-associa ed BAY Y 3118 was also s udied. PMNs we e incuba ed o 10 min a 37°C wi h BAY Y 3118 (ex acellula concen a ion, 2 mg/li e ), collec ed by cen i uga ion, and apidly suspended in quinolone- ee medium. PMN-associa ed BAY Y 3118 was quan i a ed 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 . Con ols we e always used o e alua e he e ec s o inhibi o s and subs a es on he luo escence o BAY Y 3118 in cell- ee sys ems. O ganisms and suscep ibili y es ing. S. au eus ATCC 25923 was used o killing assays. Suscep ibili y s udies we e de e - mined by dilu ion assay. The MICs and MBCs o cip o loxacin and BAY Y 3118 agains his s ain we e 0.25 and 0.5 mg/li e and 0.03 and 0.06 mg/li e , espec i ely. In acellula ac i i y o quinolones. To e alua e he in a- cellula ac i i ies o an imic obial agen s, a p e iously de- sc ibed me hod was used (12). 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) in Hanks balanced sal solu ion 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 (50 pm) o 60 min a 37°C. 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, Middlesex, Uni ed Kingdom). A ha ime (designa ed ime ze o), di e en an imic obial agen s we e added and ials we e incuba ed in a shake (50 pm) a 37°C. 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 obial agen s). Ex acellula an imic obial agen s we e emo ed by washing cells wi h ice-cold phospha e- bu e ed saline. Cells we e lysed in dis illed wa e , and samples we e dilu ed and pou pla ed on aga . Colonies we e coun ed a e 24 h o incuba ion a 37°C. 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 3 h. In addi ion o de e min- 80) E 600 a 0 o400 0 200 0 2 5 10 25 50 100 Ex acellula concen a ion (mg/1) FIG. 2. BAY Y 3118 up ake by human PMNs a di e en ex acel- lula concen a ions (n = S). Incuba ions we e ca ied ou o 20 min. E o ba s indica e s anda d de ia ions. VOL. 38, 1994 on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. E ec s o cell iabili y, en i onmen empe a u e, and pH on he in acellula pene a ion o BAY Y 3118 in human PMNs (n = 6) Exp l condi ion(s)a C/E a io Viable cells a 37°C ....................... 7.5 ± 0.9 Viable cells a 4°C ....................... 5.4 + 0.5 (P = 1.30 x 10-2)b Dead cells a 37°C ....................... 5.5 + 0.8 (P = 2.18 x 10-2)b pH 5 ....................... 8.2 1.9 pH 6 ....................... 7.6 1.9 pH 7 ....................... 7.5 1.6 pH 8 ....................... 7.5 1.9 a Expe imen s we e ca ied ou o 20 min a ex acellula concen a ions o 2 mg/li e . b Signi ican compa ed wi h iable cells a 37'C. ing 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 associa ed o ex acellula ). Samples o 50 [lI we e emo ed om bio ials and deposi ed on glass slides. A e being s ained 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 means ± s anda d de ia ions. Di e ences among g oups we e compa ed by analysis o a iance, which was used o assess s a is ical signi icance a P c 0.05. RESULTS Up ake o BAY Y 3118 by human PMNs. The up ake and e lux kine ics o BAY Y 3118 in human PMNs a e shown in Fig. 1. BAY Y 3118 pene a es in o human PMNs apidly, eaching in acellula concen a ions six o se en imes g ea e han he ex acellula ones. The e lux o BAY Y 3118 was also apid, wi h 86% o he cell-associa ed d ug being los wi hin 5 min (compa e alues a 20 and 25 min in Fig. 1). The e ec s o di e en ex acellula concen a ions on he up ake o BAY Y 3118 by PMNs a e shown in Fig. 2. The le els o he cell-associa ed d ug anged om 15.8 ± 3.2 mg/li e a an ex acellula concen a ion o 2 mg/li e (C/E a io, 7.9 + 1.6) o 655 ± 75 mg/li e a an ex acellula concen a ion o 100 mg/li e (C/E a io, 6.7 ± 1.4). The e ec s o cell iabili y, en i onmen al empe a u e, and pH on BAY Y 3118 up ake by human PMNs a e shown in Table 1. In acellula pene a ion o BAY Y 3118 in o iable PMNs was signi ican ly educed a 4°C, as was he case wi h dead cells, bu s ill eached C/E a ios o g ea e han 5. The e ec s o di e en me abolic inhibi o s on BAY Y 3118 up ake by PMNs a e shown in Table 2. Among he inhibi o s e alua ed, only sodium luo ide and sodium cyanide signi i- can ly impai ed in acellula pene a ion o his quinolone. TABLE 2. E ec s o me abolic inhibi o s on he in acellula pene a ion o BAY Y 3118 in human PMNs (n = 6)a Inhibi o C/E a io Con ol ....................... 7.5 ± 0.9 Sodium luo ide ....................... 4.3 + 2.1 (P = 4.8 x 10-2)b Sodium cyanide ....................... 2.6 + 2.0 (P = 7.9 x 10-3)b Ca bonyl cyanide m-chlo ophenyl- hyd azone ....................... 6.3 ± 1.8 2,4-Dini iphenol ....................... 5.3 ± 1.9 a Expe imen s we e ca ied ou o 20 min a ex acellula concen a ions o 2 mg/li e . b Signi ican compa ed wi h con ol. TABLE 3. E ec s o cell memb ane-s imula ing agen s on he in acellula pene a ion o BAY Y 3118 in human PMNs (n = 6)a S imulus C/E a io Con ol ............. 6.9 ± 2.3 Opsonized S. au eus ............. 4.4 ± 1.3 (P = 0.025)b Opsonized zymosan ............. 3.6 ± 1.4 (P = 1.91 x 10-1)b PMA ............. 16.6 + 8.2 (P = 1.22 x 10-2)b a Expe imen s we e ca ied ou o 20 min a ex acellula concen a ions o 2 mg/li e . b Signi ican compa ed wi h con ol. Finally, we e alua ed he in luence o phagocy osis and cell memb ane s imuli on he up ake o BAY Y 3118. We ound ha he inges ion o opsonized pa icles such as zymosan o bac e ia signi ican ly educed he up ake o BAY Y 3118 PMNs (Table 3). S imula ion wi h PMA, howe e , signi ican ly inc eased in acellula concen a ions o his d ug. In acellula ac i i y o BAY Y 3118. The in acellula ac i i y o BAY Y 3118 agains S. au eus compa ed wi h ha o cip o loxacin was e alua ed by a 3-h assay (Fig. 3). A each o h ee ex acellula concen a ions, he in acellula ac i i y o BAY Y 3118 in human PMNs was signi ican ly g ea e han ha o cip o loxacin. DISCUSSION In his s udy, he up ake o BAY Y 3118 by human PMNs was e alua ed. Using a luo ome ic assay, we obse ed ha his an imic obial agen was highly concen a ed in hese phagocy ic cells e en a he apeu ical concen a ions. The C/E a ios o BAY Y 3118 we e simila o hose obse ed o cip o loxacin, o loxacin, spa loxacin, and o he quinolones (3, 9-11). The up ake o BAY Y 3118 o PMNs was apid, nonsa - u able (a ex acellula concen a ions o be ween 2 and 200 mg/li e ), and e e sible. The in acellula pene a ion o BAY Y 3118 was a ec ed by cell iabili y, en i onmen al empe a- u e, and some me abolic inhibi o s (sodium luo ide and sodium cyanide). I is in e es ing ha e en an inhibi o o mi ochond ial oxida i e me abolism, such as sodium cyanide, dec eased he up ake o o loxacin by PMNs since hese cells ha e ew mi ochond ia and do no depend upon oxida i e me abolism o ene gy (9). This phenomenon could be ela ed 100 80 o 60 ~40 //4 Z 20/// 0.5 2 5 Ex acellula concen a ion (mg/I) FIG. 3. E ec s o BAY Y 3118 (solid ba s) and cip o loxacin (ha ched ba s) on he killing o S. au eus ATCC 25923 inges ed by human PMNs. Da a a e exp essed as pe cen ages o he le els in con ols (wi hou an imic obial agen s) (n = 5). *, P < 0.05 when compa ed wi h cip o loxacin. E o ba s indica e s anda d de ia ions. 2428 GARCIA ET AL. on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om BAY Y 3118 UPTAKE BY NEUTROPHILS 2429 o a seconda y o mino ac i i y o his inhibi o in o he me a- bolic unc ions o phagocy es. Simila esul s ha e p e iously been desc ibed o o loxacin and le o loxacin (10). The up ake o o he quinolones, such as spa loxacin, by human PMNs was no a ec ed by any o hese pa ame e s (3). Lome loxacin pene a ion and ema loxacin pene a ion in o human PMNs, howe e , we e a ec ed by en i onmen al empe a u e (11). These da a indica e ha mo e han one mechanism may media e he in acellula pene a ion o di e en quinolones. The pene a ion o BAY Y 3118 dec eased sligh ly when PMNs we e incuba ed wi h opsonized zymosan o S. au eus. The s imula ion o PMN memb anes by PMA, an ac i a o o he NADPH oxidase sys em ia p o ein kinase C (8), signi i- can ly inc eased he up ake o his quinolone by human PMNs. This di e gen e ec could be ela ed o he ac ha he ac i a ion o NADPH oxidase by opsonized pa icles di e s om ha o PMA, he o me being media ed by phospho- lipase A2 (8). The mechanism whe eby BAY Y 3118 accumu- la es in cells is no ye known, and no simple model can be p esen ed in iew o he indings p esen ed abo e. Al hough ce ain da a poin o a passi e mechanism, as has been pos ula ed o spa loxacin (3), many o he s a e ypical o an ac i e mechanism, as has been desc ibed o o loxacin and le o loxacin (10). Ou p e ious expe iences show ha al hough mos quinolones each simila in acellula concen a ions in phagocy ic cells, di e en mechanisms may be in ol ed. These mechanisms do no seem o be ela ed ei he o he deg ee o luo ina ion o he molecule (11) o o he pa i ion coe icien s (3), as has been p e iously demons a ed. A con as be ween phagocy e an imic obial agen up ake and in acellula ac i i y has been desc ibed o a ew an imi- c obial agen s (6). In ou s udy, BAY Y 3118 showed in a- cellula ac i i y agains S. au eus. In ac , his ac i i y was signi ican ly g ea e han hose o cip o loxacin and o he quinolones p e iously e alua ed (10, 11); his e ec was pa - ially due o he highe bac e icidal ac i i y o his an imic o- bial agen agains he s ain e alua ed. In summa y, BAY Y 3118 pene a es in o human PMNs, eaching in acellula concen a ions se e al imes g ea e han he ex acellula ones while emaining ac i e in acellu- la ly. The high an imic obial ac i i y o his quinolone in addi ion o he p ope ies obse ed in his s udy enhances he po en ial uses o BAY Y 3118. ACKNOWLEDGMENT We hank P. Hidalgo o p epa a ion o he manusc ip . REFERENCES 1. B emm, K. D., U. Pe e sen, K. G. Me zge , and R. Ende mann. 1992. In i o e alua ion o BAY Y 3118, a new ull-spec um luo oquinolone. Chemo he apy (Basel) 38:376-387. 2. Buggy, B. P., D. R. Schabe g, and R D. Swa z. 1984. 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