ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1993, p. 100-1050066-4804/93/010100-06$02.00/0Copyright X 1993, American Society for Microbiology

Therapeutic Effect of Cefozopran (SCE-2787), a NewParenteral Cephalosporin, against Experimental Infections

in MiceYUJI IIZAWA,* KENJI OKONOGI, RYOGO HAYASHI, TOMOYUKI IWAHI,

TOSHIYUKI YAMAZAKI, AND AKIRA IMADA

Pharmaceutical Research Laboratories III, Pharmaceutical Research Division, Takeda Chemical Industries,Ltd., 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532, Japan

Received 26 August 1992/Accepted 22 October 1992

The therapeutic effect of cefozopran (SCE-2787), a new semisynthetic parenteral cephalosporin, againstexperimental infections in mice was examined. Cefozopran was more effective than cefpiramide and was aseffective as ceftazidime and cefpirome against acute respiratory tract infections caused by Kiebsiellapneumoniae DT-S. In the model of chronic respiratory tract infection caused by K. pneumoniae 27, cefozopranwas as effective as ceftazidime. The therapeutic effect of cefozopran against urinary tract infections caused byPseudomonas aeruginosa P9 was superior to that of cefpirome and was equal to those of ceftazidime andcefclidin. In addition, cefozopran was more effective than ceftazidime and was as effective as flomoxef in a thighmuscle infection caused by methicillin-sensitive Staphylococcus aureus 308A-1. Against thigh muscle infectionscaused by methicillin-resistant S. aureus N133, cefozopran was the most effective agent. The potent therapeuticeffect of cefozopran in those experimental infections in mice suggests that it would be effective againstrespiratory tract, urinary tract, and soft tissue infections caused by a variety of gram-positive andgram-negative bacteria in humans.

The search for new drugs effective as antibacterial agentsrequires the thorough evaluation of drugs not only in vitrobut also in vivo (i.e., in the control and management ofexperimental infections). Systemic infections induced by anintraperitoneal injection of a bacterial suspension have beencommonly used for the evaluation of new antibiotics. Suchinfections, however, are not always representative of infec-tious diseases in humans. It is therefore important to evalu-ate antibiotics by using animal models of infection whichmore closely mimic the actual situation in humans.

This paper deals with the in vivo evaluation of cefozo-pran (SCE-2787), 7p-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-methoxyiminoacetamido]-3-(imidazo-[1,2-b]pyridazinium-1-yl)methyl-3-cephem-4-carboxylate, inner salt hydrochloride (Fig.1), in a variety of infection models in mice. Cefozopran is a newparenteral cephalosporin with potent in vitro activity against avariety of clinical pathogens, including Staphylococcus aureusand Pseudomonas aeruginosa (9, 17). The infection modelsused here are acute and chronic respiratory tract infectionscaused by KZebsiella pneumoniae, urinary tract infectionscaused by P. aeruginosa, and thigh muscle infections withabscess formation caused by methicillin-sensitive and -resis-tant S. aureus (MSSA and MRSA, respectively).

(This work was presented at the 29th Interscience Confer-ence on Antimicrobial Agents and Chemotherapy, Houston,Tex., 17 to 20 September 1989 [27] and the 30th InterscienceConference on Antimicrobial Agents and Chemotherapy,Atlanta, Ga., 21 to 24 October 1990 [21].)

MATERIALS AND METHODS

Antibiotics. Cefozopran (SCE-2787) (Fig. 1), cefpirome,and cefclidin were prepared in the Research and Develop-

* Corresponding author.

ment Division of Takeda Chemical Industries, Ltd. Ceftazi-dime (Nippon Glaxo Co., Ltd., Tokyo, Japan), cefpiramide(Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan), andflomoxef (Shionogi & Co., Ltd., Osaka, Japan) were ob-tained commercially. MICs of each antibiotic were deter-mined by an agar dilution method using an inoculum of ca.104 CFU and are shown in each table.Experimental infections and antibiotic treatment. (i) Respi-

ratory tract infection. Acute respiratory tract infectioncaused by K pneumoniae DT-S was induced as describedpreviously (19). Briefly, K pneumoniae DT-S was grownovernight at 37°C in brain heart infusion broth (Difco Labo-ratories, Detroit, Mich.), and cells were collected by centrif-ugation, washed with phosphate-buffered saline (Dulbeccoformula [modified] without magnesium and calcium; FlowLaboratories, Inc., McLean, Va.), and suspended in thesame buffered saline to give a suspension of ca. 109 CFU/ml.The bacterial suspension was placed in a nebulizer (Vapone-frin pocket nebulizer; USV Pharmaceutical Co., Tuckahoe,N.Y.) and aerosolized at a pressure of 1.2 kg/cm2 for 40 min.Four-week-old ICR male mice (Shizuoka Agricultural Coop-erative Associations for Laboratory Animals, Shizuoka,Japan) were used. Antibiotics were administered subcutane-ously (s.c.) twice a day for 5 days starting 20 h afterinfection. The survival rate was recorded every day until day6 after infection. The surviving mice were then killed byether inhalation, and bacterial counts in the lungs wereassessed quantitatively as described previously (19). Thedose of antibiotic (milligrams per kilogram of body weight)keeping 50% of the mice alive (50% survival dose [SD50]) andthat required for eradication of the test organisms from thelungs (less than 10 CFU/lung) in 50% of the mice tested (50%clearance dose [CD50]) were calculated by the probit method(14).Chronic respiratory tract infection caused byK pneumo-

niae 27 was induced similarly to acute respiratory tract

100

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EFFICACY OF CEFOZOPRAN AGAINST EXPERIMENTAL INFECTIONS 101

H2N> sNH~N

N CONH

N SN

OCH3 2- + *HCI

co

FIG. 1. Structure of cefozopran.

infection, as described previously (8). Five-week-old CBA/Jfemale mice (Charles River Japan, Inc., Chiba, Japan) were

exposed for 60 min to an aerosol generated from a bacterialsuspension of ca. 1010 CFU/ml. When this procedure is used,the bacterial counts in the lungs remain constant over theperiod from 1 to 4 weeks after infection (8). Antibiotics wereadministered s.c. twice a day for 7 days starting 2 weeksafter infection. Quantitative assessment of bacterial countsin the lungs was done on the day after the final treatment,and the CD50s were calculated as described above.

(ii) Urinary tract infection. Urinary tract infection causedby P. aeruginosa P9 was induced by a modification of themethod of Nishi and Tsuchiya (18). In brief, P. aeruginosaP9 was grown overnight at 37°C in King A broth (11), andcells were collected by centrifugation, washed with Trypti-case soy broth (BBL Microbiology Systems, Cockeysville,Md.), and suspended in Trypticase soy broth at a concen-tration of ca. 107 CFU/ml. CBA/J mice were anesthetized byintraperitoneal injection of 50 mg of sodium pentobarbital

per kg of body weight. After the mice were forced to voidurine by compression of the bladder through the externalabdominal wall, 0.05 ml of the bacterial suspension was

transurethrally inoculated into the bladder. Then, the exter-nal urethral meatus was clamped for 6 h. Antibiotics wereadministered s.c. twice a day for 5 days starting 3 days afterinfection. Mice were killed by cervical dislocation the dayafter the final treatment. The kidneys were removed asepti-cally and homogenized with 4 ml of Trypticase soy broth,and the bacterial count in the homogenate was determined.The CD5Os were calculated by the probit method on the basisof the number of mice in which the kidneys were sterilized(less than 20 CFU per pair of kidneys).

(iii) Thigh muscle infection. A modification of the abscessmodel described by Selbie and Simon (23) was used. Cells(ca. 5 x 107 CFU) of S. aureus 308A-1 (MSSA) grownovernight at 37°C in brain heart infusion broth were injectedinto the left thigh muscles of ICR mice which were anesthe-tized with 62.5 mg of sodium pentobarbital per kg. Antibiot-ics were administered s.c. 2, 18, and 26 h after infection, andmice were killed by cervical dislocation on day 5. Theinfected thigh muscle was aseptically removed and homog-enized in 3 ml of Trypticase soy broth, and the bacterialcount in the homogenate was determined. Treatment wasjudged effective if the bacterial count decreased to 1/10 themean value in the control mice or less. The dose effective in50% of the mice tested (50% effective dose) was calculatedby the probit method. Similar experiments were done withMRSA N133. The bacterial count in the thigh muscle was

examined on day 7 after infection with MRSA, and treatmentwas judged effective if the bacterial count decreased to 1/100the mean value in the control mice or less. In both infections,

TABLE 1. Therapeutic effect of cefozopran and reference compounds against acute respiratory tract infections caused byKpneumoniae DT-S in micea

Drug and dose (mg/kg)' Survival ratec SD50 (mg/kg)d Clearance ratee CD50 (mg/kg)d MIC (>g/ml)

None 0 of 10 0 of 10

Cefozopran5 OoflO 0 of 1020 8 of 10 13.8 (7.80-23.8) 2 of 10 29.0 (16.8-51.0) 0.0580 10 of 10 10 of 10

Ceftazidime5 1 of 10 0 of 1020 10 of 10 9.02 (6.69-12.5) 1 of 10 36.0 (26.7-49.9) 0.02580 10 oflO 10 of 10

Cefpiramide20 0 of 10 116f(67-204) 0 of 10 >320f 0.280 2 of 10 0 of 10320 10 of 10 1 of 10

Cefpirome5 2of 10 1 of 1020 10 of 10 7.25 (4.21-12.8) 5 of 10 23.6 (10.4-47.9) 0.02580 10 of 10 8 of 10320 10 of 10 10 of 10

a Infection was induced as described in the text, and bacterial recovery immediately after infection was (3.4 + 1.2) x 103 CFU per lung (mean ± standarddeviation).

b Antibiotics were administered s.c. twice a day for 5 days. Dose is expressed as amount of antibiotic per injection.c Survival rate on day 6 after infection.d SD50 and CD50 were calculated from the survival rate and the clearance rate, respectively. Values in parentheses indicate 95% confidence limits.e Bacterial counts in the lungs were determined on day 6 after infection. Clearance rate is expressed as the number of mice in which the lungs were sterilized

per total number treated.f Significantly different from the value for cefozopran (P < 0.05).

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TABLE 2. Therapeutic effect of cefozopran and ceftazidimeagainst chronic respiratory tract infections caused by

K pneumoniae 27 in micea

Drug and(g) ratec CD50 (mg/kg)' MIC (,ug/ml)

None 0 of 10

Cefozopran20 1 of 1240 0 of 11 55.2 (48.8-62.9) 0.0580 12 of 12

Ceftazidime20 0 of 1240 1 of 11 62.8 (47.7-82.6) 0.180 9 of 12160 11 of 11

a Infection was induced as described in the text, and bacterial recoveryimmediately after infection was (6.0 + 4.4) x 105 CFU per lung (mean ±standard deviation).

b Antibiotics were administered s.c. twice a day for 7 days. Dose isexpressed as amount of antibiotic per injection.

I Bacterial counts in the lungs were determined the day after the finalantibiotic treatment. Clearance rate is expressed as the number of mice inwhich the lungs were sterilized per total number treated.

I CD50 was calculated from the clearance rate. Values in parenthesesindicate 95% confidence limits.

the minimum detectable count was 103 CFU per thighmuscle.

Statistics. The difference between the efficacy of cefozo-pran and those of other antibiotics was analyzed by astandard parallel-line bioassay procedure (1). In all cases,differences were considered significant at P < 0.05.

RESULTSEfficacy against respiratory tract infection. (i) Acute infec-

tion caused by K. pneumoniae DT-S. All of the untreatedcontrol mice died around 3 days after infection. Most of themice that received 20 mg of cefozopran, ceftazidime, orcefpirome per kg of body weight survived, but most of themice that received 80 mg of cefpiramide per kg of bodyweight died (Table 1). The SD50 of cefozopran was 13.8mg/kg of body weight, which was much lower than that ofcefpiramide (116 mg/kg) and equal to those of ceftazidime(9.02 mg/kg) and cefpirome (7.25 mg/kg). Cefozopran wasalso highly effective for eradicating the infecting organismfrom the lungs, as were ceftazidime and cefpirome; completeeradication was seen in all mice that received 80 mg or moreof cefozopran per kg (Table 1). However, cefpiramide wasnot able to eradicate the bacteria even at the highest dose(320 mg/kg). The efficacy of cefozopran for eradication, asestimated by the CD50, was superior to that of cefpiramideand was equal to that of each of the other antibiotics usedhere.

(ii) Chronic infection caused by K. pneumoniae 27. Table 2shows that cefozopran was effective in chronic respiratorytract infections in which K pneumoniae 27 introduced bythe aerosol method was maintained at a level of 105 to 106CFU per lung over the period from 1 to 4 weeks afterinfection (8). The test organism was eradicated from thelungs in all mice treated with 80 mg of cefozopran per kgtwice a day for 7 days starting 2 weeks after infection.Ceftazidime did not completely eradicate the organism at thesame dose. The CD50 values of cefozopran and ceftazidime,however, were not significantly different.

Efficacy against urinary tract infection caused by P. aeru-

ginosa P9. The therapeutic effects of cefozopran and refer-ence compounds on urinary tract infections in mice werecompared. The bacterial count in the kidneys of untreatedmice was 105 CFU on day 8 when mice were killed forobservation. Cefozopran, at a dose of 6.25 mg/kg, eradicatedP. aeruginosa from the kidneys of half of the infected mice(Table 3). The therapeutic effect of cefozopran was compa-rable to those of ceftazidime and cefclidin and was signifi-cantly superior to that of cefpirome.

Efficacy against thigh muscle infection. (i) Infection causedby MSSA. The efficacy of cefozopran was assessed by usingthigh muscle infection in mice. MSSA 308A-1 cells injectedinto the thigh muscle of ICR mice were maintained for atleast 5 days at 106 CFU. The organism was resistant toantibiotic treatment and was not eradicated completely evenat the highest dose of any antibiotic tested. The number oforganisms at the infection site, however, was reduced dosedependently by all antibiotics tested. Therefore, we judgedtreatment effective if the antibiotic eradicated 90% or moreof the bacteria from the infection site. Three antibioticsexhibited a therapeutic effect parallel to their in vitro anti-bacterial activity (Table 4). Cefozopran was superior toceftazidime and comparable to flomoxef.

(ii) Infection caused by MRSA. Similar thigh muscle infec-tion was established with MRSA N133. Antibiotic treatmentwas judged effective if 99% or more of the bacteria were

TABLE 3. Therapeutic effect of cefozopran and referencecompounds against urinary tract infections caused by

P. aeruginosa P9 in mice'

Drug ando Clearance ratec CD50 (mg/kg)' MIC (jig/ml)

None 0 of 15

Cefozopran1.56 3 of 156.25 7 of 15 8.78 (2.86-21.4) 0.7825 11 of 15100 12 of 15

Ceftazidime1.56 0 of 106.25 6 of 10 14.6 (4.85-49.9) 1.5625 5 of 10100 8 of 10

Cefpirome1.56 0 of 106.25 2 of 10 40.8e (16.1-330) 6.2525 5 of 10100 6 of 10

Cefclidin1.56 1 of 106.25 4 of 10 18.4 (5.48-106) 0.7825 6 of 10100 7 of 10a Infection was induced as described in the text."Antibiotics were administered s.c. twice a day for 5 days. Dose is

expressed as amount of antibiotic per injection.c Bacterial counts in the kidneys were determined the day after the final

antibiotic treatment. Clearance rate is expressed as the number of mice inwhich the kidneys were sterilized per total number treated.

d CD50 was calculated from the clearance rate. Values in parenthesesindicate 95% confidence limits.

' Significantly different from the value for cefozopran (P < 0.05).

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TABLE 4. Therapeutic effect of cefozopran and referencecompounds against thigh muscle infections caused by

MSSA 308A-1 in micea

Drug and dose Bacterial recovery' ED50 MIC(mg/kg) No. of bacteria Effective rate (mg/kg)" (pg/ml)

None 6.59 ± 0.48 0 of 10

Cefozopran0.625 6.14 1.42 2 of 82.5 5.38 ± 1.12 3 of 8 2.87 (0.75-7.56) 0.3910 4.77 1.19 6of840 4.59 + 0.96 8of8

Ceftazidime10 6.45 0.26 Oof840 5.29 ± 1.09 4 of 8 56.1e (20.7-135) 6.25160 4.35 0.95 7of8640 4.06 1.14 7of 8

Flomoxef0.625 5.83 ± 0.64 2of82.5 5.54 ± 0.70 5 of 8 1.73 (0.12-5.08) 0.3910 4.34 ± 1.20 7of840 3.37 ± 1.08 7 of 8

a Infection was induced as described in the text.bAntibiotics were administered s.c. 2, 18, and 26 h after infection. Dose is

expressed as amount of antibiotic per injection.c Bacterial counts in the thigh muscle were determined on day 5 after

infection (log CFU; mean ± standard deviation). Bacterial counts less than103 CFU were regarded as 103 CFU to calculate the mean. Effective rate isexpressed as the number of mice in which treatment was judged effective pertotal number treated.

d ED50, 50% effective dose, was calculated from the effective rate. Valuesin parentheses indicate 95% confidence limits.

e Significantly different from the value for cefozopran (P < 0.05).

eradicated from the infection site on day 7 after infection,because infection was maintained for at least 7 days and thebacterial counts were reduced markedly by the therapy.Cefozopran was more effective than ceftazidime and was

slightly more effective than flomoxef (Table 5). Cefozopranexerted the strongest activity in reducing the bacterial count,especially at the highest dose (200 mg/kg).

DISCUSSIONCefozopran, a new semisynthetic cephalosporin, showed

a potent therapeutic effect against all infections used in thisstudy. The pharmacokinetic profile of cefozopran is knownto be similar to those of reference antibiotics used in thisstudy, except for the protein-binding rate of cefpiramide inmouse serum (10, 12, 15).Acute respiratory tract infection caused by K pneumo-

niae has been used frequently to investigate the effects ofantibiotics against pneumonia (2-4, 13, 16, 20), because Kpneumoniae is a well-known pathogen in community-ac-quired bacterial pneumonia and the experimental infection isinduced easily in mice by the aerosol method (19). Againstthe infection, cefpiramide was less effective than we hadexpected by the MIC than other antibiotics. The plasmahalf-life of cefpiramide after intravenous administration inmice is reported to be 11 min, which is comparable to thoseof other antibiotics, including cefozopran (12, 15). However,the binding of cefpiramide to serum protein in mice is 44%,which is higher than that of cefozopran (7.1%) (12, 15). Thismight result in the marked difference in effectiveness of thetwo antibiotics. We have also used a model of chronicrespiratory tract infection caused byK pneumoniae. Sincechronic infections in humans are difficult to treat success-fully, it is valuable to examine the efficacy of an antibiotic inan experimental chronic infection. In this model, lobarconsolidation occurs primarily in the anterior and middlelobes of the right lung and in the median lobe beginning day

TABLE 5. Therapeutic effect of cefozopran and reference compounds against thigh muscle infections caused by MRSA N133 in micea

Drug and dose Bacterial recovery' ED g/kg)d MIC (pg/ml)(mg/kg)" No. of bacteria Effective rate

None 8.18 + 0.23 0 of 10

Cefozopran3.125 7.63 ±0.41 0 of 812.5 7.55 + 0.43 0 of 8 79.8 (39.4-181.7) 5050 6.54 0.95 2 of 8200 4.82 ± 1.29 7 of 8

Ceftazidime3.125 7.93 ± 0.57 Oof812.5 7.88 ± 0.40 Oof8 >200w >10050 7.61 ± 0.65 Oof8200 6.99 0.64 lof8

Flomoxef3.125 7.86 ±0.39 Oof 812.5 7.16 + 0.47 0 of 8 116 (56-417) 10050 7.18 0.67 lof 8200 5.86 0.95 6 of 8

a Infection was induced as described in the text."Antibiotics were administered s.c. 2, 18, and 26 h after infection. Dose is expressed as amount of antibiotic per injection.c Bacterial counts in the thigh muscle were determined on day 7 after infection (log CFU; mean + standard deviation). Bacterial counts less than 103 CFU were

regarded as 103 CFU to calculate the mean. Effective rate is expressed as the number of mice in which treatment was judged effective per total number treated.dED50, 50% effective dose, was calculated from the effective rate. Values in parentheses indicate 95% confidence limits.Significantly different from the value for cefozopran (P < 0.05).

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3 after infection (8). The bacterial counts in the lungs remainat a constant level over the period from 1 to 4 weeks afterinfection despite sufficient production of specific antibodyagainst the bacteria. Only antibiotics showing good penetra-tion into the inflammatory sites and potent bactericidalactivity have a therapeutic effect in this model, which isconsidered to be very useful when predicting the clinicaleffectiveness of antibiotics. Cefozopran was effective in bothacute and chronic respiratary tract i-nfection models. Theseresults suggest that cefozopran would be useful in treatingcomplicated chronic respiratory tract infections as well asacute pneumonia.

P. aeruginosa is one of the major pathogens responsiblefor nosocomial infections, and the urinary tract is a commonsite of P. aeruginosa infections in humans (26). Therefore,we induced an ascending urinary tract infection with P.aeruginosa and evaluated the efficacy of cefozopran againstthe infection. Among the antibiotics used in this study,cefozopran showed the highest level of activity against theinfection.Mouse thigh muscle has been frequently used as an

infection site when examining the efficacy and pharmacoki-netics of antibiotics (4-7, 22, 24, 25). The thigh muscleinfection caused by S. aureus used here is a persistentinfection with abscess formation. This infection model isconsidered suitable to predict the effect of antibiotics againstinfections of skin and soft tissue. Cefozopran showed ther-apeutic activity superior to or similar to that of the referenceantibiotics in both MSSA and MRSA infections.The fundamental strategy of chemotherapy for infectious

diseases is to decrease the bacterial count to some level; theremaining organisms are usually eliminated by the hostdefense factors. However, infections often occur in immu-nocompromised hosts who do not have the ability to elimi-nate even a small number of pathogens. In these cases,antibiotics with strong bactericidal activity are necessary.Cefozopran is known to possess strong bactericidal activityagainst a variety of pathogens (17). In fact, cefozopran waseffective in experimental chronic respiratory tract infectionin which the host defense was not working well, indicatingthat it can be expected to exert therapeutic activity even inimmunocompromised hosts.

Infections caused by S. aureus and P. aeruginosa arepredominant problems in clinical situations. The resultspresented here demonstrate that cefozopran affords goodcoverage against infections in which S. aureus and P.aeruginosa can be expected. This new cephalosporin shouldtherefore be useful for the treatment of local infections inhumans.

ACKNOWLEDGMENTS

We thank N. Kitamoto and J. Obita for technical assistance.

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