Bioevaluation of the Antibacterial Flumequine for Urinary Tract Use

Transcription

1 ANTIMICROBIAL AGENTS AND CHZMOTHEXRY, JUIy 1976, p Copyright C 1976 Americn Society for Microbiology Vol. 10, No. 1 Printed in U.S.A. Bioevlution of the Antibcteril Flumequine for Urinry Trct Use S. R. ROHLFING,* J. F. GERSTER, AND D. C. KVAM Riker Lbortories, Inc., St. Pul, Minnesot Received for publiction 17 Februry 1976 The ntimicrobil ctivity of flumequine (R-802) ws chrcterized by in vitro nd in vivo procedures. Assy of the miniml inhibitory concentrtions for 321 recent clinicl isoltes reveled tht 88% of the grm-negtive bcteri were inhibited by n R-802 concentrtion of 6.2,ug/ml or less. Cross-resistnce in lbortory-derived mutnts ofproteus vulgris ws essentilly complete for R- 802, nlidixic cid, nd oxolinic cid, lthough quntittive differences were evident. R-802 ws more effective thn either of these quinolone ntibcterils in preventing the development of experimentl murine pyelonephritis (P. vulgris). R-802 nd trimethoprim/sulfmethoxzole (1:5) were eqully effective in resolving P. mirbilis-induced prosttitis of rts. Flumequine (R-802) is synthetic ntibcteril, selected from series of tricyclic quinolines prepred in Riker Lbortories. The compound is 6,7-dihydro-9-fluoro-5-methyl-1-oxo- 1H,5H (U) quinolizine-2-crboxylic cid (Fig. 1). This report describes the bioevlution of R-802 nd its comprison with severl ntimicrobils in clinicl use. (This work ws reported in prt t the Annul Meeting of the Federtion of Americn Societies for Experimentl Biology, 1974, Atlntic City, N.J.) pensions. The conditions of incubtion were overnight t 37 C in humidified tmosphere enriched with 10% crbon dioxide. Growth inhibition of those strins tht ppered to be most susceptible to R-802 ws further exmined by log2 dilution tube technique in nutrient broth (5). Nlidixic cid nd oxolinic cid were included in this titrtion s reference quinolone ntibcterils. Bcillus subtilis ws deleted in this comprison becuse of pellicle formtion in sttic broth culture; Proteus vulgris, which ws not tested on TSA becuse of the "swrming" phenomenon, ws included here. The miniml inhibitory concentrtions (MICs) of R-802 for clinicl isoltes of urinry trct infection were similrly MATERIALS AND METHODS determined; the influence of culture medium ph ws exmined in selected strins t 0.2-unit intervls Bcteri. Lbortory stock cultures were used. over the rnge 6.0 to 8.0. Susceptibility testing of clinicl isoltes ws ccomplished with cultures from urine specimens tken t growth on oxolinic cid, nlidixic cid, nd R-802 Cross-resistnce of mutnt clones selected by the St. Pul Veterns Administrtion Hospitl, St. ws ssessed by the replic plting technique (6). Pul, Minn. These isoltes were identified using Approximtely 109 cells were spred over the surfce conventionl microbiologicl methods (1). Wound of drug pltes contining TSA plus 0.15% bile slts isoltes of Stphylococcus ureus were obtined to inhibit swrming ofp. vulgris 210. The levels of from the St. Louis Prk Clinic, Minnepolis. drug permitting development of pproximtely 10 Antimicrobils. Flumequine (R-802) ws synthesized in Riker Reserch Lbortories. Stock solu- cid, 10,ig/ml; nlidixic cid, 50,ug/ml. No colonies colonies/plte were s follows: R-802 nd oxolinic tions of R-802 (1%) for in vitro use were prepred were observed on pltes contining 25,ug of R-802 or with 0.1 N sodium hydroxide. Nlidixic cid nd oxolinic cid per ml. An imprint of the colonies oxolinic cid were provided by Winthrop Lbortories nd Wrner-Lmbert Reserch Institute, re- sterile velveteen templtes. These in turn were used developing on ech originl plte ws trnsferred to spectively. Bctrim (sulfmethoxzole nd trimethoprim, 5:1), Erythrocin (erythromycin sterte), nd tining the individul drugs. All pltes were scored to reproduce the ptterns on severl test pltes con- Terrmycin (oxytetrcyline-hydrochloride) were obtined in commercil form. Efficcy. Preclinicl evlution of R-802 for fter 2 dys of incubtion t 37 C. Susceptibility. The ntimicrobil spectrum of R- tretment of urinry trct infection used rt 802 ws surveyed on tryptone soy gr (TSA) pltes model, which mimics the development of bcteril with log,0 dilution series. Microliter liquots of pyelonephritis in humns (8). One-tenth milliliter of stock solution were mixed with molten TSA in individul screw-cp vils nd dispensed into petri injected into the prtilly ligted bldder of femle n overnight shke culture of P. vulgris 210 ws dishes. These preprtions were llowed to hrden Simonsen rts. Tryptone soy broth ws used s the nd were surfce inoculted with stock culture sus- growth medium nd diluent. The temperture of 20

2 VOL. 10, 1976 F BIOEVALUATION OF THE ANTIBACTERIAL FLUMEQUINE 21 OH FIG. 1. Structure offlumequine (R-802). incubtion ws 35 to 37 C with rotry gittion t 200 rpm. The highest dilution (usully 1:10) producing uniform bldder infections, which persisted for t lest 10 dys, ws used. Antimicrobils were suspended in 4% queous cci nd homogenized with ground-glss tissue mill. Treted nimls were dosed dily by orl gvge beginning 24 h postinfection for period of 3 weeks. Tretment periods of only 10 dys t elevted doses (nmely, 100 mg of R-802/kg per dy) were indequte bsed on culture of excised tissue. Infection control nimls received no tretment. Wter ws supplied d libitum. Animls were scrificed 24 h post-tretment to culture both kidneys nd the urinry bldder. Kidney slices nd bldder frgments were plced on TSA gr to revel the chrcteristic swrming outgrowth in positive cultures. No ntibcteril ctivity could be demonstrted in excised tissues by using sensitive indictor bcteri. Infection relpse ws ssessed by retining nimls for period of 2 weeks post-tretment prior to scrifice nd culture. An experimentl prosttitis ws induced in mle Simonsen rts ccording to the method of Friedlnder nd Brude (4). The growth conditions for P. mirbilis 120 were the sme s those described forp. vulgris 210. The undiluted culture in nonrefluxing volume (0.05 ml) ws injected into the unrestricted bldder of surgiclly prepred nimls. Antimicrobils were suspended in 4% queous cci nd homogenized with ground-glss tissue mill. Treted nimls were dosed dily by orl gvge beginning 5 dys postinfection for period of 3 weeks. Infection control nimls received no tretment. Wter ws supplied d libitum. Animls were scrificed fter totl of 25 dys to culture the ventrl prostte, the urinry bldder, nd both kidneys on TSA pltes; the dorsolterl prostte is less frequently involved nd ws not monitored for evidence of infection. Urinry levels of ctive drug were estimted by gr diffusion ssy using Klebsiell pneumonie 113 s the indictor bcterium. In lieu of cylinders, stinless-steel templte contining six wells (biossy plte no. 1310, Lbline Instruments, Inc.) ws plced on the surfce of seeded TSA pltes. R-802 stndrds nd pooled urine smples were diluted in tryptone soy broth. Ech rt (three per group) received single dose of 10 or 25 mg of R-802 per kg prior to 24-h urine collection. RESULTS In vitro. The susceptibility to R-802 of selected strins of microorgnisms is presented in Tble 1. The concentrtions of R-802 inhibiting growth of bcteri rnged from 1 to 100 jig/ml, wheres no ntifungl ctivity ws evident t 100,ug of R-802 per ml. Antibcteril ctivity ws unchnged by the ddition of 10% (vol/vol) horse serum to the TSA growth medium. The MICs of quinolone ntibcterils presented in Tble 2 suggested tht study of the response of recent clinicl isoltes from urinry trct infection to R-802 should be pursued. No "skip-tube" end points indicting rpid selection of resistnt progeny were evident in this experiment. Assy of the R-802 MICs for 321 recent clinicl isoltes (Tble 3) reveled tht 88% of the grm-negtive bcteri were inhibited t concentrtion of 6.2 ug/ml or less. The mjority of grm-positive strins exhibited n MIC of 12.5,ug/ml or more. A different response to R-802 by these two groups of bcteri ws lso suggested by the influence of growth medium ph on ntimicrobil ctivity in broth culture. The MIC vlues for Escherichi coli VA-1, P. vulgris TABLE 1. Microorgnism Antimicrobil ctivity ofr-802 MIC (jg/ml) Serum firee 10% Horse serum Streptococcus pyogenes Stphylococcus ureus Bcillus subtilis Escherichi coli Pseudomons eruginos Streptococcus feclis Klebsiell pneumonie Aspergillus niger 18 >100 >100 Cndid lbicns 20 >100 >100 Growth inhibition of lbortory stock cultures determined by TSA plte dilution. TABLE 2. MICs of quinolone ntibcterils MIC (zgiml) Bcterium Nli- Oxodixic linic R-802 cid cid Proteus vulgris Klebsiell pneumonie Escherichi coli Stphylococcus ureus Growth inhibition of lbortory stock cultures determined by the seril dilution tube technique in nutrient broth.

3 22 ROHLFING, GERSTER, AND KVAM TABLE 3. Cumultive percentge of clinicl isoltes inhibited t vrious R-802 concentrtions Bcterium MIC (,g/ml): Totl >25 ltes Escherichi coli b Proteus vulgris Proteus mirbilis Proteus rettgeri Proteus morgnii Klebsiell pneumonie Enterobcter erogenes Pseudomons eruginos Serrti mrcewcens Providence Streptococcus feclis Stphylococcus ureuse _ 6 Growth inhibition determined by the seril dilution tube technique in nutrient broth. b _, Zero incidence. c Wound isoltes. VA-1, nd P. mirbilis VA-3 were invrint in the ph rnge 6.0 to 8.0; the MIC for S. ureus 148 incresed 10-fold to mximum of 25.0 ug/ ml t ph 8.0. Cross-resistnce in lbortory-derived mutnts ofp. vulgris ws essentilly complete for R-802, nlidixic cid, nd oxolinic cid (Tble 4). Although discrete colonies were evident on nlidixic cid selection pltes contining 10 ug of drug per ml, bckground lwn of dditionl mutnts ws confirmed on replic pltes. Multistep mutnts selected by seril trnsfers in incresing concentrtions of ech drug confirmed these observtions: cross-resistnce ws demonstrted for P. vulgris clones cpble of growing on TSA contining 100,ug of R-802, 100,ug of oxolinic cid, or 500 Mg of nlidixic cid per ml. In vivo. the R-802 dose response obtined in n scending rt urinry trct infection model is presented in Tble 5. The medin effective dose bsed on severl titrtions in this model ws pproximtely 10 mg/kg per dy. The dt were not cumultively scored s the sum of ll positive cultures for ech dose level, becuse upon cesstion of tretment nimls with infected bldders would surely develop infected kidneys in due course. A vrible number of nimls succumbed to ftl infections prior to terminting the experiment, s indicted in the column of survivors. The comprtive efficcy of R-802 nd two urinry trct ntimicrobils is presented in Tble 6. The performnce of nlidixic cid ws comprble to tht of oxolinic cid, wheres R- 802 ws clerly more effective in preventing the development ofpyelonephritis s defined in this model. Bldder isoltes from the two R-802 tretment filures were indistinguishble from the infecting culture in terms of ntimicrobil susceptibility. The bsence of infection relpse in shortterm follow-up experiment is evident in Tble 7. Bldder isoltes from the four rts treted TABLE 4. Cross-resistnce ofp. vulgris clones isolted in the presence of certin quinolone ntib-,cterils Originl selection plte Cross-resistnt clones on test replic plte QuOino- Concn NofOxomglnc NLidixic cid R-802 lone m)colonies Acid (10 g 50jg/ ml ml) jsg/ml) ~ml ml ml Oxolinic cid Nlidixic TNTC TNTC 4 cid TNTC 9 4 R TNTC, Too numerous to count. TABLE 5. ANTIMICROB. AGENTS CHEMOTHER. Ascending rt urinry trct infection model: titrtion of R-802 SriosLeft Right Group (21dys) kid- kid- Bldder Group 2 )ney ney I Infection controls (P. 8/10 6/8 6/8 7/8 vulgris) Infected nd treted with R-802 (mg/kg per dy) /10 0/10 0/10 1/ /10 0/10 0/10 0/ /10 0/10 0/10 0/ /10 0/10 0/10 0/ /10 2/9 1/9 4/9 Ech group consisted of 10 nimls; the lst three columns indicte the rtio of positive cultures to survivors. TABLE 6. Ascending rt urinry trct infection model: comprison of R-802 with reference quinolones Survivors Left Right Group (21 dys) kid- kid- Bldder ney ney Infection controls (P. 8/10 7/8 7/8 7/8 vulgris) Infected nd treted with: R-802, 25 mg/kg per 10/10 0/10 0/10 2/10 dy Nlidixic cid, 25 9/10 2/9 4/9 4/9 mg/kg per dy Oxolinic cid, 25 9/10 3/9 4/9 6/9 mg/kg per dy Ech group consisted of 10 nimls; the lst three columns indicte the rtio of positive cultures to survivors.

4 VOL. 10, 1976 with 25 mg of R-802/kg per dy were gin unchnged in terms of R-802 susceptibility. The therpeutic response to R-802 nd three reference ntimicrobils in P. mirbilis-induced prosttitis is presented in Tble 8. In three experiments, the incidence of positive prostte cultures from infection control groups of 10 nimls ech rnged from 0.72 to 0.86; positive cultures from both kidneys of ech niml closely prlleled the incidence of prostte infection. The incidence of bldder infection ws slightly higher, s indicted. R-802 ws essentilly ineffective in this model t dose of TABLE 7. Ascending rt urinry trct infection model: infection incidence 14 dys post-tretment1 Time Sur- Left Right Group (dys) vivuors kid- kid- Bldder Group dys) ivorsney ney Infection controls 21 5/10 4/5 4/5 4/5 (P. vulgris) Infected nd treted with R- 802 (mg/kg per dy) /7 0/7 0/7 0/ (+14) 7/8 0/7 0/7 0/ /8 2/6 1/6 3/ (+14) 6/7 1/6 0/6 1/6 The infection control group consisted of 10 nimls; 15 nimls were treted in ech dosge group s indicted. The lst three columns indicte the rtio of positive cultures to survivors. BIOEVALUATION OF THE ANTIBACTERIAL FLUMEQUINE mg/kg per dy. A dose of 50 mg/kg per dy resulted in 65% reduction in the incidence of prosttitis nd pyelonephritis, s mesured by in vitro culture of excised tissue. Orl dministrtion of erythromycin nd trimethoprim/sulfmethoxzole significntly reduced the incidence of positive prostte cultures t 50 nd 100 mg/kg per dy. These reductions were not closely prlleled by results obtined from tissues ofthe urinry trct per se. Oxytetrcycline ws poorly effective in this model by comprison with the other ntimicrobils exmined. Bsed on the in vitro ctivity of R-802, the levels of ctive drug chieved in 24-h urine collection from rts receiving single orl dose were 15 jig/ml t 10 mg of R-802 per kg of body weight nd 41,ug/ml t 25 mg of R-802 per kg of body weight. DISCUSSION Quinolone ntimicrobils re brod-spectrum gents reserved primrily for urinry trct use. Therpeutic dvntges for Proteus sp. infections, in prticulr, were suggested in comprtive study of 2,081 clinicl isoltes which used Neg-grm sensitivity disks (9). In one study of urinry trct infections, nlidixic cid performed significntly better thn sulfdimidine, producing cure rtes of 93.7 nd 81.4%, respectively; this result led the uthors to regrd nlidixic cid s drug of choice when bcteriologicl fcilities re not vilble (2). It is lso cler tht development of resistnt orgnisms fter dministrtion of existing quinolones is frequent problem (3,7). There is n TABLE 8. Efficcy ofr-802 nd reference ntimicrobils in P. mirbilis-induced prosttitis Tretment Survivors Prostte Bldder Right kidney Left kidney Infection controls 24/30 19/24 (0.79) 22/24 (0.92) 20/24 (0.83) 20/24 (0.83) Infected nd treted with R-802 (mg/kg per dy) 25 10/10 7/10 (0.70) 8/10 (0.80) 8/10 (0.80) 8/10 (0.80) 50 29/30 8/29 (0.28) 16/29 (0.55) 9/29 (0.31) 9/29 (0.31) /10 0/10 0/10 0/10 0/10 Erythromycin (mg/kg per dy) 50 7/10 1/7 (0.14) 6/7 (0.86) 6/7 (0.86) 6/7 (0.86) 100 8/10 2/8 (0.25) 6/8 (0.75) 4/8 (0.50) 2/8 (0.25) Oxytetrcycline (mg/kg per dy) 50 10/10 5/10 (0.50) 6/10 (0.60) 6/10 (0.60) 6/10 (0.60) 100 9/10 4/9 (0.44) 7/9 (0.78) 6/9 (0.67) 6/9 (0.67) Trimethoprim/sulfmethoxzole (mg/kg per dy) 50 8/10 3/8 (0.38) 8/8 (1.00) 7/8 (0.88) 6/8 (0.75) /10 1/10 (0.10) 6/10 (0.60) 2/10 (0.20) 4/10 (0.40) Survivl rtios indicte the number of nimls tht succumbed to infection between dy 5, when tretment ws initited, nd dy 25, when specimens were tken. The lst four columns indicte the rtio of positive cultures to survivors; the incidence of infection clculted from this rtio is shown in prentheses.

5 24 ROHLFING, GERSTER, AND KVAM pprent bsence of trnsferble drug resistnce with these compounds, due in prt to the presrbed indictions. Lbortory-induced muttions to quinolone resistnce occur in stepwise fshion, nd the synthesis of oxolinic cid tht enhnced in vitro ctivity improved the potentil therpeutic vlue of these compounds. The results obtined in the present study utilizing pproprite rodent models demonstrte the dded potentil of R-802. In vitro testing ofr-802 demonstrted significnt ntibcteril ctivity for the principl pthogens in urinry trct infection. In ddition to E. coli, Proteus sp., nd the Kkebsiell- Enterobcter group, the clinicl isoltes exmined in this study lso included 32 Serrti mrcescens; S. mrcescens is usully regrded s n index of nosocomil infection nd ppered to resemble E. coli in terms of R-802 susceptibility. The pronounced influence of ph on ntibcteril ctivity versus S. ureus hs been observed with other quinolones nd is unexplined t present. In vivo testing of R1802 ws imed t determining efficcy for intended use. The Proteus sp.-induced pyelonephritis is good model for compring quinolone ntibcterils for resons previously cited. It is less useful for ntibiotics, e.g., mpicillin, but ttempts to substitute other infecting bcteri hve met with limited success. Prosttitis cn be significnt tretment problem nd source of chronic bcteruri (10). The rodent model used to chllenge R-802 for this indiction is idelized in terms of the infecting gent; nevertheless, the method provides mesure of ntimicrobil efficcy rther thn ntimicrobil ctivity prtitioned into prosttic secretions. The reference ntimicrobils in this experiment were selected lrgely on the bsis of clinicl interest. Oxytetrcycline hydrochloride would be poor choice bsed on in vitro susceptibility of most strins ofp. mirbilis (9). The in vitro ctivity of erythromycin sterte is fvored by slightly lkline conditions, nd the ph of mmmlin prosttic fluid ANTimicRoB. AGzNTs C*MMOTHZR. is usully less thn 7.0. Nevertheless, erythromyc ws effective in eliminting beri from the te. the peutic ponses to R1402 nd trimethoprim/suifmetox ole were dose relted nd comprble for prosttitis. Additionl idictions for 4-02 hve been investigted in this lbortory nd will be reported in seprte communiction. ACKNOWLEDGMENTS We exprs our ppretion to J. Cherny, St. Pul Veterns Administrtion Hospitl, who collected nd identified clinicl isoltes usd in this study. Technicl ssistnce ws provided by P. L. Bettermn, G. K. Hill, nd R. M. Winndy. Chemicl technology ws provided by S. Pes core nd R. Stern. LITERATURE CITED 1. Blir, J. E., E. H. Lennette, nd J. P. Trunt (ed.) Mnul of clinicl microbiology, p Americn Society for Microbiology, Bethesd, Md. 2. Brown, P. C. C., M. C. Denwhy, P. H. DeetOR, D. H. M. Titcombe, nd D. M. McLen Sulphdimidine nd nlidixic cid therpy in urinry-trct infections in generl prctice. Prctitioner 207: Clrk, H., N. K Brown, J. F. Wllce, nd M. Turek Emergence of resistnt orgnisms s function of done in oxolinic cid therpy. Am. J. Med. Sci. 261: Friedlnder, A. M., nd. L Brude Experimentl prosttitis.reltionship to pyelonephritis. J. Inflit. Dis. 125: Grove, D. C., nd W. A. Rndll Assy methods of ntibiotics, lbortory mnul, p Medicl Encyclopedi, Inc., New York. 6. L, J., nd E. M. Lederberg Replic plting nd indirect selection of bcteril mutnts. J. Bcteriol. 63: Mdsen, P. O., nd P. R. Rhodes Oxolinic cid, new chemotherpeutic gent in the tretment of urnnry trct infection. J. Urol. 105: Mirgli, G. J Model infections in the evlution of ntimicrobil gents. Trns. N.Y. Acd. Sci. 32: Roup, A., nd J. D. Pguet Snsibilit6 ux ntibiotiques de 2081 souches bct6riennes, isoldes d'infections urinires. Schweiz. Med. Wochenchr. 98: Stmey, T. A Prt II: the clinicl spects of urinry trct infections-observtions on the clinicl use of nlidixic cid. Postgrd. Med. J. 47:21-26.