ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1978, p. 137-145 0066-4804/78/1302-0137$02.00/0 Copyright i) 1978 American Society for Microbiology
Vol. 13, No. 2 Printed in U.S.A.
SCE-129, Antipseudomonal Cephalosporin: In Vitro and In Vivo Antibacterial Activities KANJI TSUCHIYA, MASAHIRO KONDO,* AND HIROSHI NAGATOMO Central Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan Received for publication 31 August 1977 SCE-129
[3-(4-carbamoyl-1-pyridiniomethyl)-7,8-(D-a-sulfophenylacetamido)-
ceph-3-em-4-carboxylate monosodium salt], a new semisynthetic cephalosporin, shows potent in vitro antibacterial activities against Pseudomonas aeruginosa and some gram-positive bacteria, whereas it shows lower activity against many gram-negative rods. Against clinical isolates of P. aeruginosa this cephalosporin exhibited higher activity than did carbenicillin, and against the strains of Staphylococcus aureus, SCE-129 had similar activity to carbenicillin. Variations in pH, addition of horse serum, and type of growth medium had no significant effects on the activity of the cephalosporin; however, the inoculum size had some effect on the activity. SCE-129 is an effective bactericidal agent against P. aeruginosa and S. aureus. The protective effects of SCE-129 in mice infected with P. aeruginosa and S. aureus were more potent than those of carbenicillin. The protective effects of SCE-129 on Pseudomonas infection in mice varied according to the dosage schedule and the challenge dose. In a multiple dose schedule, a smaller amount of SCE-129 was necessary than that in a single dose schedule. The effects of SCE129 after subcutaneous or intraperitoneal administration were more potent than were those by intravenous administration. No protective effect was observed by oral administration.
SCE-129 (CGP 7174/E) [3-(4-carbamoyl-1-py-
ridiniomethyl)-7,8-(D-a-sulfophenylacetamido)ceph-3-em-4-carboxylate monosodium salt] (Fig. 1) is a new semisynthetic cephalosporin which is selected from a series of 7-sulfophenyl-acetamido cephalosporins. It is a compound developed as a result of collaborative research between Takeda Chemical Industries, Ltd. and Ciba-Geigy Ltd. and was synthetized by Takeda Chemical Industries, Ltd. The relationships between chemical structures and microbiological activity of these cephalosporins were reported by Nomura et al. (11-13). The present report describes the in vitro and in vivo antibacterial activities of SCE-129 with carbenicillin as a reference compound. MATERIALS AND METHODS Antibiotics. SCE-129 was prepared by Takeda Chemical Industries, Ltd. Carbenicillin (Gripenin) was purchased from Fujisawa Pharmaceutical Co., and penicillin G (Penicillin G Kalium Takeda) was obtained from a commercial source. Bacteria. Laboratory strains were maintained on Trypticase soy agar (TSA) (BBL) or TSA supplemented with a 10% bovine blood slant (blood-TSA). Clinical isolates of Pseudomonas aeruginosa and Staphylococcus aureus were kindly supplied by S. Mitsuhashi, Gunma University and Y. Shimizu, the Central Clinical Laboratory; Osaka University Hospi-
tal, and were maintained on Dorset egg medium at
40C until use.
Determination of MIC. Minimum inhibitory concentration (MIC) was determined by the agar dilution method. One loopful (2 mm in diameter) of a suspension containing about 10' colony forming units (CFU)/ml or 106 CFU/ml, cultivated overnight on TSA, blood-TSA, or Tryticase soy broth (TSB) (BBL), was streaked for a length of about 2 cm on TSA, blood-TSA, or MacConkey agar (Eiken), or in TSB containing twofold serial dilutions of each antibiotic. The MIC was defined as the lowest concentration of antibiotic that prevented visible growth after overnight incubation at 37°C. Determination of MBC. Each tube of TSB containing twofold serial dilutions of each antibiotic was inoculated with test organisms to obtain a concentration of about 106 CFU/ml. After overnight incubation at 37°C, the MIC was obtained after inspecting the tubes for turbidity. A 2-pl portion (calibrated loop) from each tube was subcultured onto an antibioticfree TSA plate. The MBC was defined as the lowest concentration of antibiotic preventing visual growth on the subculture plate. Development of resistance in vitro. To generate resistance, the organisms were grown in 5 ml of TSB in the presence of twofold serial dilutions of each antibiotic. From the tube containing the highest concentration of the antibiotic that allowed normal or nearly normal growth, successive transfers were made every 48 h into the next series of tubes containing the same and higher concentrations. 137
138
TSUCHIYA, KONDO, AND NAGATOMO Hm-CO-NH
S_ 03.4o
o>'
2 3Co-"M2 42
FIG. 1. Chemical structure of SCE-129.
Bactericidal activity. Each TSB containing twofold serial dilutions of an antibiotic was inoculated with test organisms to obtain a concentration of about 106 CFU/ml. Control tubes contained no antibiotic. After incubation at 370C for 0, 2, 4, 6 and 8 h, portions from these cultures were plated on TSA. After 24 h of incubation, the number of colonies on each TSA plate was counted. Measurement of macromolecular substances in cells. S. aureus FDA 209 P was grown in TSB to the half-maximum growth. The cells were then harvested and divided into four portions: two were treated with SCE-129 to give concentrations of 1 and 10 tig/ml, respectively, one was treated with penicillin G to give a concentration of 0.05 Lg/ml, and the last one was used as a control. To monitor cell growth, the optical density of the bacterial suspension was measured at 650 nm by a Coleman Universal spectrophotometer. Portions of the culture were centrifuged, and the cells were washed twice with water. The protein fraction of each organism was isolated as described by Park male SLC-ddY mice weighing 19 to 23 g were infected of Lowry et al. (7). N-Acetylaminosugar was extracted and determined by the method of Reissig et al. (15). Nucleic acids from S. aureus were isolated according to the method of Schneider (17). Deoxyribonucleic acid was determined by the diphenylamine method and ribonucleic acid was determined by the orcinol method. Protective test. P. aeruginosa and S. aureus were cultured overnight in King A broth and brain heart infusion (Difco), respectively. The cultures were suspended in 5% mucin (Laboratories Division of Wilson Pharmaceutical and Chemical Co.). Four-week-old male SLC-ddY mice weighing 19 to 23 g were infected intraperitoneally with the organism suspended in 0.5 ml of 5% mucin. The challenge doses of P. aeruginosa and S. aureus were respectively about 100 times and 30 times the number of organisms required to kill 50% of the challenge control mice. Groups of five mice at each dose level were subcutaneously given 0.2 ml of antibiotics by following the schedule given in the table. All experiments were repeated four to five times. The 50% effective dose (ED50, mg/kg) was calculated by provit analysis from the survival rates recorded 7 days after infection (6).
ANTIMICROB. AGENTS CHEMOTHER.
aeruginosa and S. aureu& The antibacterial activities of SCE-129 and carbenicillin are illustrated in Fig. 2 and 3. Against 119 strains of P. aeruginosa, SCE-129 was about 10 times as active as carbenicillin. With the inoculum of 108 CFU/ml, 104 of 119 (87.4%) strains were inhibited at 1.56 to 25 utg of SCE-129 per ml, whereas only 1 of 119 (0.8%) strains was inhibited at 25 ,ug of carbenicillin per ml. Carbenicillin-resistant strains that were resistant to 400 jig of the penicillin per ml were susceptible to SCE-129 at 100 ug or less per ml. A similar pattern was observed on 106 CFU/ml as inoculum suspension. Against 112 strains of S. aureus, about 90% of which were resistant to penicillin G, SCE-129 showed activity similar to that of carbenicillin on iOW CFU/ml of inoculum suspension. With the inoculum of 101 CFU/ml, carbenicillin was more potent than SCE-129. Effects of inoculum size, medium pH, horse serum, and culture medium. The 50-SCE
4
-129
40
30-
0
m
II I
.
I
20
E
I
M
10
I114I /Il II
II/ I/
I
0.M 3C132.5
50gm200
4
I In I
0.
0.78 113 12.5
400
.50
200 . 400
MIC in pg/mi FIG. 2. Susceptibility of 119 clinically isolated strains of P. aeruginosa to SCE-129 and carbenicillin. MIC in' pg/ml
110
110
lOVmI
~~~~~~06.,,
100/m
100 90
90
-SCE-129 70
-70-
60
L_50
I
60
50o
40E40 RESULTS Z 30 oa 30 Antibacterial spectrum. SCE-129 was / found to be a unique antibiotic because the 20 o S S 20 4 ,/ 4 activity of SCE-129 was confined to P. aeruginosa and some gram-positive bacteria. It was 0 0 practically inactive against other gram-negative 0 39 1.56 6.22 25 0.39 1.56 6.25 25 rods. Various strains of P. aeruginosa showed MIC in IC in- pg/mi M pg/mi higher susceptibility to SCE-129 than to carbenicillin (Table 1). FIG. 3. Susceptibility of 112 clinically isolated Activity against clinical isolates of P. strains of S. aureus to SCE-129 and carbenicillin. I
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
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139
TABLE 1. Antibacterial spectrum of SCE-129 and carbenicillin MIC (ag/ml)
Mediumb
Organisma
SCE-129
Pseudomonas aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa
Escherichia coli
SP U 31 N 18 D 363 P8 NIHJ JC-1 Umezawa K-12 0-78 0-111 0-143 DT A
TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA MCA MCA MCA MCA MCA
6.25 6.25 3.13 6.25 50 50 100 50 50 50 50 50 50 100 50 25 25 50 50 50 100 50 >100 >100 100 100 >100 100 >100 >100 100 3.13 6.25 6.25 3.13 1.56 3.13 3.13 25 25 6.25 6.25 1.56 100
Carbenicillin 50 100 3.13 100 100 12.5 6.25 25 6.25 1.56 3.13 6.25 50 6.25 12.5 6.25 6.25 12.5 3.13 12.5 12.5 3.13 3.13 1.56 6.25 1.56 50 1.56 6.25 0.78 3.13 0.39 0.78 12.5 0.2 0.2 0.2 0.2 1.56 0.39 0.2 0.2 1.56 0.39
E. coli E. coli E. coli E. coli E. coli Klebsiellapneumoniae Salmonella paratyphi S. schottmuelleri S. hirschfeldii S. typhi Boxhill-58 S. typhi Watson S. typhimurium Shigella dysenteriae EW-1 S. flexneri EW-10 S. flexneri EW-40 S. sonnei EW-33 Proteus mirabilis IFO 3849 P. mirabilis IFO 12255 P. vulgaris IFO 3851 P. vulgaris IFO 3988 P. vulgaris OX-19 P. vulgaris OX-K MCA P. morganii IFO 3168 MCA P. morganii IFO 3848 MCA Vibrio cholerae Inaba MCA FDA 209 P MCA Staphylococcus aureus S. aureus 308 A-1 MCA S. aureus 1840C MCA Streptococcus pyogenes E-14 Blood-TSA S. pyogenes Dick Blood-TSA S. pyogenes S-8 Blood-TSA S. pyogenes NY-5 Blood-TSA S. mitior America Blood-TSA S. pneumoniae type I Blood-TSA S. pneumoniae type II Blood-TSA S. pneumoniae type III Blood-TSA Corynebacterium diphtheriae Tronto Blood-TSA Bacillus subtilis PCI 219 Blood-TSA a Inoculum size: One loopful of bacterial suspension (10k CFU/ml). b TSA, Trypticase soy agar (BBL); Blood-TSA, TSA supplemented with 10% bovine blood; MCA, MacConkey agar (Eiken). c S. aureus 1840, Penicillin G-resistant strain.
MICs of SCE-129 against P. aeruginosa U 31 and D 363 and S. aureus FDA 209 P were determined under various conditions. The activity of SCE-129 increased as the inoculum size decreased. The MIC of SCE-129 was lower in an acidic pH than in an alkaline pH. The addition of 10 to 50% horse serum to the medium slightly increased the antibacterial activity of SCE-129. The activity of SCE-129 was not af-
fected by varying the culture medium (Table 2). Development of resistance in vitro. The resistance of P. aeruginosa U 31 to SCE-129 and carbenicilin was rapidly acquired. After the 4th transfer, the MICs of both antibiotics exceeded 1,600 ,ug/ml. Resistance of S. aureus to SCE-129 was acquired at a slightly slower rate than to carbenicillin. At the 23rd transfer, the
140
ANTimicRoB. AGENTS CHEMOTHER.
TSUCHIYA, KONDO, AND NAGATOMO
TABLE 2. Effect of various factors on the antibacterial activity of SCE-129 and carbenicillin MIC (jg/mi)
Carbeniciljin
SCE-129 Factor P. aeruginosa
Inoculum sizea 103 104 106 106 107 108 Medium pHb 6 7 8 9 Horse serum (%)C 0 10 20 50 Mediumd TSA NA MH HI BHI
S.
P. aerugmosa
aurew
aurew
U 31
D 363
0.78 1.56 1.56 3.13 3.13 6.25
25 50 50 50 100 200
25 25 25 50 50 100
0.05 0.1 0.2 0.2 0.39 0.78
1.56 3.13 3.13 6.25
3.13 3.13 6.25
6.25
100 100 200 200
25 50 100 100
0.39 0.39 0.78 0.78.
12.5 6.25 6.25 6.25
6.25 3.13 1.56 1.56
6.25 3.13 3.13 3.13
200 200 100 100
100 100 50 50
1.6 0.78 0.78 Q.78
6.25 6.25 6.25 6.25 6.25
3.13 3.13 3.13 3.13 6.25
U 31
D 363
1.56 3.13 3.13 6.25 6.25 12.5
0.78 1.56 1.56 1.56 1.56 3.13
6.25 6.25 12.5 12.5
FDA29P
.
FDAO9P
3.13 100 50 0.39 3.13 200 100 0.39 3.13 200 100 0.39 200 3.13 100 0.39 200 3.13 100 0.39 Inoculum site (CFU/ml): One loopful of bacterial suspension; Medium: TSA. b Inoculum sise: One loopful of bacterial suspension (106 CFU/ml); Medium: TSA. c Inoculum size: 0.1 ml of bacterial suspension (106 CFU/ml); Medium: TSB. d Inoculum size: One loopful of bacterial suspension (106 CFU/ml); Medium: TSA, Trypticase soy agar (BBL); NA, nutrient agar (Eiken); MH, Mueller-Hinton medium (Eiken); HI, heart infusion agar (Eiken); BHI, brain heart infusion agar (Eiken).
MIC of SCE-129 reached 400 itg/ml and, up to the 36th transfer, the MIC value did. not increase. The MIC of carbenicillin exceeded 400 ,ug/ml at the 16th transfer (Fig. 4). Cross resistance. The presence of cross resistance among SCE-129, carbenicillin, and four other cephalosporins was studied with highly resistant strains, which developed in vitro, of P. aeruginosa U 31 and S. aureus FDA 209 P. A cross resistance was observed between SCE-129 and carbenicillin in P. aeruginosa, and als among SCE-129, carbenicillin, and cephalexin in S. aureus, whereas the other three cephalosporins showed somewhat different patterns of cross resistance to S. aurezs (Table 3). Relationships between MIC and MBC. The MICs of SCE-129 determined by the. tube dilution assay closely resembled the MBCs against 9 strains of P. aeruginosa and 10 strains of S. aureus. The MIC and the MBC of SCE129 against P. aeruginosa were apparently lower than those of carbenicillin (Table 4). Bactericidal activity. SCE-129 was an effec-
S. aureus FDA 209 P
P. aeruginosa U 31
- - icdhl
1600
ICmb..icilli.
400-
I
100'
I
i400 -/ C
!
SCU-129
/
100
CE-12 6.25.
N
2
r
4e
Number of Transfer
I
i
10
is
20
25
,0 .5
Number of Transfer
FIG. 4. Patterns of development of resistance ofP. aeruginosa U 31 and S. aureus FDA 209 P to SCE129 and cqrbenicillin.
tive bactericidal agent against P. aeruguiosa and S. aureus. An apparent decrease in the number of living bacteria was observed at a concentration of 6.25 1g/ml for P. aerugnosa U 31 and 3.13 uAg/ml for S. aureus FDA 209- P, the concentrations being their MICs determiined by the tube dilution assay, and similar kiUl curves were observed at concentrations higher than 12.5 and 6.25 pg/ml, respectively (Fig. 5). Effect on the synthesis of macromolecu-
VOL. 13, 1978
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
141
TABLE 3. Cross resistance among SCE-129 and related antibiotics' MICb (Ag/Mi)
Organism
CB-PC
P. aeruginosa U 31 P. aeruginosa P. aeruginosa
(parent) r-CB-PC r-SCE-129
200 >1,600 >1,600
SCE-129
CER
CET
CEZ
CEX
25 200 >1,600
S. aureus FDA 209 P (parent) 0.39 3.13 0.05 0.1 0.2 1.56 S. aureus r-CB-PC >400 200 100 200 400 >400 S. aureus r-SCE-129 50 200 0.78 3.13 6.25 400 S. aureus r-CER 3.13 3.13 400 200 50 >400 S. aureus r-CET 25 12.5 100 400 400 >400 S. aureus r-CEZ 25 25 12.5 100 400 >400 S. aureus r-CEX 25 12.5 3.13 25 100 >400 a Inoculum size: One loopful of bacterial suspension (10' CFU/ml). Medium: TSA. b Antibiotics: CB-PC, carbenicdilin; CER, cephaloridine; CET, cephalothin; CEZ, cefazolin; CEX, cephalexin. TABLE 4. MIC and MBC of SCE-129 and carbenicillin for clinically isolated P. aeruginosa and S. aureus SCE-129 Carbenicillin MIC MIC MBC MBC Organism (pg/mi) (pg/mi) (jug/ni) (lg/mi) P. aeruginosa GN-3007-T 6.25 12.5 200 200 P. aeruginosa GN-3310-T 12.5 12.5 200 400 P. aeruginosa GN-3324-T 12.5 12.5 200 400 P. aeruginosa GN-3325-T 12.5 12.5 200 400 P. aeruginosa GN-3327-T 12.5 12.5 200 400 GN-3333-T 12.5 P. aeruginosa 12.5 200 400 GN-3338-T P. aeruginosa 12.5 12.5 400 400 GN-3363-T P. aeruginosa 6.25 6.25 50 100 P. aeruginosa GN-3365-T 6.25 6.25 100 100
S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus
T-3 T-15 T-20 T-30 T-32 T-62 T-63 T-69 T-78 T-88
6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.26 6.25
6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25
0.78 3 13 3.13 0.78 6.25 3.13 3.13 3.13 6.25 3.13
0.78 3.13 3.13 0.78 6.25 3.13 6.25 3.13 6.25 3.13
lar substances. The addition of 10 ,ug of SCE- cephalosporin administration. No protective ef129 or 0.05 ,ug of penicillin G per ml to a culture fect of SCE-129 was observed in a single 400of S. aureus resulted in growth suppression, but mg/kg subcutaneous dose 0, 1, 2, or 4 h after the growth of S. aureus was not affected by 1 infection (Table 5). As the frequency of the ,ug of SCE-129 per ml. The maxmum accumu- cephalosporin administration increased, the prolation of N-acetylaminosugar was found after 40 tective effect was enhanced. The protective efto 80 min of incubation with 10 ytg of SCE-129 fect was similarly potent in the groups treated or 0.05 ug of penicillin G per ml. The synthesis 0 and 2 h, 0 and 4 h, and 1 and 4 h after infection. of cellular RNA, DNA, and protein was not The effect in the triple dose was superior to significantly inhibited after 40 min of incubation that in the double doses; the dosage schedule with the indicated concentration of antibiotics of 0, 2, and 4 h after infection produced the most potent activity among the triple doses. (Fig. 6). Protective effects of various dosage Furthermore, four doses, given 0, 1, 2, and 4 h schedules. The effect of SCE-129 in mice in- after infection, showed the most potent protecfected with P. aeruginosa U 31 was markedly tive effect. From the above results, the further influenced by the frequency and interval of the evaluation of the protective effect of SCE-129
142
ANTIMICROB. AGENTS CHEMOTHER.
TSUCHIYA, KONDO, AND NAGATOMO
S. aureus FDA 209 P
P. aeruginosa U 31 10*
9. 8~
f
/
7.
o
jig /MI
0.78
pg/mI
1'.1.56 pg/ml .
cm
6-
C c
5
._
0 -
4,
3.13
Vk
6
,g/mi
25jp
jig/ml
3
:.>
2
K6 12.5ugm/ 25 jig/mi I
6
/m
i
i
Hours Hours U P. SCE-129 on aeruginosa 31 and S. aureus FDA 209 P. FIG. 5. Bactericidal effect of N-Ac.tyl-
Growth
1 .0
DNA
RNA
Protein
aminosugar
0.5S 0.3
0.6
0.6
0.2
0.4
0.4.
0.1
0.2
0.2
0.4-
a C a 0.6.
0.3.
U ._
0.4-
0.2-
0
0.2-
0.1.
4\
0.0.
0.0. 40
120
0.0*
0.0 40
120
40
120
40
120
40
120
Minutes FIG. 6. Effect of SCE-129 on growth, RNA, DNA, protein, and N. acetylaminosugar synthesis in S. aureus , SCE-129, 10 pg/ml; ------, 1 pg/mi; - - -, FDA 209 P. Symbols: -, Control; PCG, 0.05 pg/ml.
in P. aeruginosa-infected mice was performed by the dosage schedule 0, 2, and 4 h after infection.
Effect of challenge doses. The protective effect of SCE-129 in mice infected with various challenge doses of P. aeruginosa was studied (Table 6). P. aeruginosa NC-5 was about 100,000 tines more virulent than P. aeruginosa U 31. However, a similar protective effect was observed in mice infected with the miniimum number of organisms required to kill all untreated mice (1 MLD). In mice infected with P. aeruginosa NC-5 whose challenge dose was increased from 1 MLD (101 CFU/mouse) to 100 MLD
(103 CFU/mouse), the EDro values changed from 9.67 to 27.6 mg/kg, and further increase of the challenge dose produced a remarkable increase of ED5o values. The remarkable increase of ED5o values was observed in mice infected with a relatively low virulent strain, P. aeruginosa U 31. Comparative protective effects of SCE129 and carbenicillin. The protective effects of SCE-129 and carbenicillin were compared in mice infected with P. aeruginosa and S. aureus (Tables 7 and 8). P. aeruginosa N 18 was very susceptible to carbenicillin. Although MICs of both antibiotics against this strain were the
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
VOL. 13, 1978
TABLE 5. Effect of dosage schedule on ED50 of SCE129 against P. aeruginosa U 31 infection in mice2 Treatment (h) 0
1 2 4 0
+ 1
0+2 0+4 1+2 1+4 2+4
0+1+2 0+ 1+4 0+2+4 1+2+4
ED60 (mg/kg)b
>400 >400 >400 >400 >400 201 194 >400 243 >400
143
in mice SCE-129 was four times more potent than carbenicillin. In mice infected with P. aeruginosa D 363, SP, NC-5, and U 31, which show usual susceptibility against carbenicillin, SCE-129 was remarkably effective compared with carbenicillin. The protective effects of both same,
TABLE 6. Effect of challenge dose on ED60 of SCE129 against P. aeruginosa infection in mice4 ChalEDMm (mg/kg)b
(112-735) (153-258)
lenge
(173-424)
(CFU
j(al)
258 (178-546) 104 (81.2-134) 75.0 (58.8-99.5) >300
U 31 P. aeruginosa (MIC, 6.25 pg/ml)
NC-5 P. aerugnosa (MIC, 6.25 pg/ml)
101
9.67 (6.02-13.6) 19.9 (15.7-24.9) 27.6 (23.5-32.2) 48.4 (39.6-61.6) i05 207 (155-326) 10" 1,840 (1,320-3,410) 17.2 (13.8-21.8) 60.1 (49.5-77.5) >2,400 107 10 >2,400 >2,400 aMice were injected intraperitoneally with indicated CFU of test organism in 0.5 ml of 5% mucin. SCE-129 was administered subcutaneously 0, 2, and 4 h after infection. bED0 values were calculated as a total dose. Numbers in parenthess indicate 95% confidence limits.
102 103 104
0+1+2+4 51.0 (37.5-71.7) a MIC of SCE-129 against the organism was 6.25 jg/ml. Mice were injected intraperitoneally with 107 CFU of test organism per animal in 0.5 ml of 5% mucin. SCE-129 was administered subcutaneously. I ED80 values were calculated as a total dose. Numbers in parentheses indicate 95% confidence limits.
TABLE 7. Comparative protective effect of SCE-129 and carbenicillin on P. aeruginosa infection in mice4 P. aeruginosa
Antibioticb Antibiotic'
~
Challenge dose
(CFU/ainima) 106
MIC
Do(gk) D0(gk)
(gi
3.13 3.13
3.43 (2.80-4.13) 15.9 (13.2-19.2)
106
6.25 50
9.14 (7.08-11.4) 504 (408-615)
SCE-129 Carbenicillin
103
6.25 100
21.0 (17.5-25.4) 1,210 (1,040-1,420)
SCE-129 Carbencillin
107
6.25 100
60.1 (49.5-77.5) 1,720 (1,430-2,180)
N 18
SCE-129 CarbenicilHin
SP
SCE-129 Carbenicillin
NC-5 U 31
10" 438 (307-633) 6.25 SCE-129 100 >2,400 Carbenicillin a Mice were injected intraperitoneally with test organism in 0.5 ml of 5% mucin. b Antibiotics were administered subcutaneously 0, 2, and 4 h after infection. c ED50 values were calculated as a total dose. Numbers in parentheses indicate 95% confidence limits. D 363
TABLE 8. Comparative protective effect of SCE-129 and carbenicillin on S. aureus infection in micea AniitcChallenge MIC S. aureus Antibiotic EDro (mg/kg)b strain (g/niml) doC l 6.25 SCE-129 9.45 (7.16-12.5)" 10" 308 A-i
Carbenicillin
0.78 aMice were injected intraperitoneally with test organism in 0.5 ml of 5% mucin. b Antibiotics were administered subcutaneously 0 h after infection. c Numbers in parentheses indicate 95% confidence limits.
10.5 (8.13-13.8)
144
TSUCHIYA, KONDO, AND NAGATOMO
ANTIMICROB. AGENTS CHEMOTHER.
antibiotics in D 363-infected mice were weaker than those in mice infected with other strains, although MICs of D 363 to SCE-129 and carbenicillin, were similar in these strains. In vitro SCE-129 showed less activity against S. aureus 308 A-1 than did carbenicillin, but in mice SCE-129 was more potent than carbenicillin. Effect of administration route. SCE-129 was similarly effective in subcutaneous and intraperitoneal routes in mice infected with P. aeruginosa U 31. It is supposed that SCE-129 was well absorbed from the injection site. By the intravenous route the effect of SCE-129 was weaker. It may depend on a more rapid decrease of the plasma level. No protective effects were observed by the oral dose of cephalosporin (Table 9).
was not observed with SCE-129 at the concentration tested. It has been reported that cephalosporins, like penicillins, are inhibitors of cell-wall synthesis (1-5, 16). The accumulation of N-acetylaminosugar in the cells of S. aureus treated with SCE129 was observed at the concentration of 10 ,ug/ml, which did not affect the synthesis of DNA, RNA, and protein. It is suggested, therefore, that SCE-129 is an inhibitor of cell-wall synthesis of S. aureus. Bacteriolytic action of SCE-129 was observed with S. aureus but not with P. aeruginosa (unpublished data). Several cephalosporins showed bacteriolytic action against E. coli. Cephalosporin 7/30 [7-(S-benzylthioacetamido)-cephen-3-
DISCUSSION Against P. aeruginosa, SCE-129 was about 10 times more active than carbenicillin. The activity of SCE-129 against carbenicillin-resistant strains of P. aeruginosa was inferior to that against carbenicilhin-susceptible strains. The moderate activity of SCE-129 against carbenicillin-resistant P. aeruginosa may be attributed to the stability of SCE-129 against hydrolysis by /8-lactamase from carbencilin-resistant P. aeruginosa. The Vmax of SCE-129, which is expressed as the relative rate of hydrolysis, assuming that the rate for penicillin G was 100, was 0.2 for,B-lactamase from carbenicillin-susceptible strains of P. aeruginosa U 31 and 13 for ,Blactamase from carbenicillin-resistant strains of P. aeruginosa GN 3407. The Km of SCE-129 against the former ,B-lactamase was 192 mM, and the Km against the latterfl-lactamase was 7,100 mM (K. Okonogi et al., personal communication). Though several cephalosporins showed the so-called "paradox phenomenon" on bactericidal action against S. aureus (18), this phenomenon TABLE 9. Effect of administration route on ED50 of SCE-129 against P. aeruginosa infection in mice" Administration EDso (mg/kg)b route
65.1 (55.1-77.0) Subcutaneous ......... 59.6 (46.7-76.8) Intraperitoneal ......... Intravenous ......... 101 (80.8-129.8) Oral ......... >1,200 a Mice were injected intraperitoneally with 107 CFU of test organism per animal in 0.5 ml of 5% mucin. SCE-129 was administered 0, 2, and 4 h after infection. b ED5o values were calculated as a total dose. Numbers in parentheses indicate 95% confidence limits.
ylmethyl-N-dimethyldithiocarbamate-4-carboxylic acid)] inhibited the growth of E. coli, but did not reveal the bacteriolytic action (5). It is also reported that cephalosporin 7/30 inhibited the protein synthesis of E. coli (4). The mode of action of SCE-129 against P. aeruginosa needs further investigation. SCE-129 showed a potent protective effect in mice infected with P. aeruginosa. In vitro activity of SCE-129 was stronger than that of carbenicillin, and in vivo SCE-129 was frequently much more active than would be expected from its activity in vitro. For example, in vitro susceptibility of P. aeruginosa N 18 against SCE-129 was the same as that against carbenicillin, but in the protective test in N 18-infected mice, SCE-129 was four times more active than carbenicillin. In mice infected with P. aeruginosa SP or NC-5, SCE-129 was about 60 times more active, but only 16 times more active in vitro. In mice infected with S. aureus, SCE-129 was more active than carbenicillin, although in vitro activity of SCE-129 against this strain was less than that of carbenicillin. There are many reports concerning the effect of dosage schedule on the experimental murine infection. MacLeod and Stone (8) showed that the therapy by sulfonamide on pneumococcus type I infection in mice must be continued for a period of 48 to 72 h for recovery, but a considerable proportion of the animals survived by treatment with penicillin in triply divided doses at 4-h intervals on the day of infection. Zubrod (19) showed that in the treatment with multiple doses of penicillin G or penicillin K hemolytic streptococcus infection in mice, survival of the mice depends on the total dose administered over a fairly broad range of dosage schedules. Baker and Jackson (18) also reported that in streptococcal infection in mice, administering daily penicillin dosage in a series of small and equally spaced doses was not more effective than administering an equivalent amount in 1 or 2
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
VOL. 13, 1978
doses per day. In typhoid infection in mice, Miller et al. (9) showed that the double dosage of antibiotic in mice challenged with S. aureus and Salmonella schotmulleri was most effective and that by increasing the frequency of treatment, the total amount of antibiotic increased. The studies reported here indicate that in the treatment of acute intraperitoneal Pseudomonas infection in mice, the amount of SCE-129 necessary to protect varied by the dosage schedule. Little or no protective effect was observed after the doses at 0 and 1 h, 1 and 2 h, or 2 and 4 h postinfection compared with that of a single dose, but the protective effect of SCE-129 was observed on other double dosage schedules. A more potent effect was observed during the multiple dosage schedule. From the results indicated here, it seems that the administration at 0 h after infection is necessary to exert the potent effect. The effect of SCE-129 reflects the relationship between the cephalosporin concentration in the body fluid and the length of time this antibiotic sustained a high MIC value. The results with SCE-129 treatment in mice may not be directly transferrable to humans. Nevertheless, it is clear that these potent effects may be seen more frequently in multiple dosages under well controlled schedules rather than in a large single dose. ACKNOWLEDGMENT We are indebted to T. Iwahi and S. Shili for their technical asistance.
LITERATURE CITED 1. Bond, J. M., R. W. Brimblecomb, and R. C. Codner. 1962. Biological assay of cephalosporin C. J. Gen. Microbiol. 27:11-19. 2. Chang, T.-W., and L Weinstein. 1964. Morphological changes in Gram-negative bacilli exposed to cephalo. thin. J. Bacteriol. 88:1790-1797. 3. Chang, T.-W., and L Weinstein. 1964. Inhibition of synthesis of the cell wall of Staphylococcus aureus by cephalothin. Science 143:807-808. 4. Fountain, ILH., and A. D. Rusele. 1969. Studies on the mode of action of some cephalosporin derivatives. J. Appl. Bacteriol. 32:312-321. 5. Gonnella, J. S. 1967. In vitro and clinical effect of cephaloridine: a semisynthetic antibiotic. Am. J. Med. Sci.
145
254:93-104. 6. Litchffeld, J. T., and F. Wilcoxon. 1949. A simple method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96:99-113. 7. Lowry, O. H., J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 8. MacLead, C. H., and E. R. Stone. 1945. Differences in the nature of antibacterial action of the sulfonamides and penicillin and their relation to therapy. Bull. N.Y. Acad. Med. 21:375-388. 9. Miller, A. K., D. L Wilmer, and W. F. Verwey. 1950. Effect of penicillin dosage schedule on treatment of experimental typhoid in mice. Proc. Soc. Exp. Biol. Med. 74:62-64. 10. Miller, A. K, and H. Kropp. 1967. Antibacterial chemotherapy tests. II. Influence of route and number of treatments on in vivo effectiveness of streptomycin and tetracycline and influence of resistance to rechallenge, p. 377-381. Antimicrob. Agents Chemother. 1966. 11. Nomura, H., T. Fugono, T. Hitaka, I. Minami, T. Azuma, S. Morimoto, and T. Masuda. 1974. Semisynthetic ,8-lactam antibiotics. 6. Sulfocephalosporins and their antipseudomonal activities. J. Med. Chem. 17:1321-1315. -12. Nomura, H., T. Fugono, T. Hitaka, I. Minami, T. Azwna, S. Morimoto, and T. Masuda. 1974. Semisynthetic ,44actam antibiotics. VII. New semisynthetic cephalosporinsr derived from a-sulfophenylacetic acid. Heterocycles 2:67-72. 13. Nomura, H., I. Minami, T. Hitaka, and T. Fugono. 1976. Semisynthetic ,B-lactam antibiotics. 8. Structureactivity relationships of a-sulfocephalosporins, J. Antibiot. 29:928-936. 14. Park, J. T., and R. Hancock. 1960. A fraction procedure for studies of the synthesis of cell-wall mucopeptide and of other polymers in cells of Staphylococcus aureus. J. Gen. Microbiol. 22:249-258. 15. Reisig, J, L, J. L Stromiuger, and L F. Leloir. 1955. A modified colorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217:959-966. 16. Russell, A. D,, and R. H. Fountain. 1971. Aspects of the mechanism of action of some cephalosporins. J. Bacteriol. 106:65-69. 17. Schneider, W. C. 1957. Determination of nucleic acid in tissues by pentose analysis. Methods Enzymol. 3:680-684. 18. Tsuchiya, K., T. Nishi, T. Oishi, S. Goto, and Y. Kaneko. 1976. Antibacterial activity of cephacetrile. Chemotherapy (Tokyo) 24:1-15. 19. White, E. J., M. J. Baker, and E. R. Jackson. 1948. Therapeutic effectiveness of single and divided dose of penicillin in a streptococcal infection in mice. Proc. Soc. Exp. BioL Med. 67:199-203. 20. Zubrod, C. G. 1947. Comparative efficiency of single and multiple dosage regiments of the penicillins. Bull. Johns Hopkins Hosp. 81:400-410.
Vol. 13, No. 2 Printed in U.S.A.
SCE-129, Antipseudomonal Cephalosporin: In Vitro and In Vivo Antibacterial Activities KANJI TSUCHIYA, MASAHIRO KONDO,* AND HIROSHI NAGATOMO Central Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan Received for publication 31 August 1977 SCE-129
[3-(4-carbamoyl-1-pyridiniomethyl)-7,8-(D-a-sulfophenylacetamido)-
ceph-3-em-4-carboxylate monosodium salt], a new semisynthetic cephalosporin, shows potent in vitro antibacterial activities against Pseudomonas aeruginosa and some gram-positive bacteria, whereas it shows lower activity against many gram-negative rods. Against clinical isolates of P. aeruginosa this cephalosporin exhibited higher activity than did carbenicillin, and against the strains of Staphylococcus aureus, SCE-129 had similar activity to carbenicillin. Variations in pH, addition of horse serum, and type of growth medium had no significant effects on the activity of the cephalosporin; however, the inoculum size had some effect on the activity. SCE-129 is an effective bactericidal agent against P. aeruginosa and S. aureus. The protective effects of SCE-129 in mice infected with P. aeruginosa and S. aureus were more potent than those of carbenicillin. The protective effects of SCE-129 on Pseudomonas infection in mice varied according to the dosage schedule and the challenge dose. In a multiple dose schedule, a smaller amount of SCE-129 was necessary than that in a single dose schedule. The effects of SCE129 after subcutaneous or intraperitoneal administration were more potent than were those by intravenous administration. No protective effect was observed by oral administration.
SCE-129 (CGP 7174/E) [3-(4-carbamoyl-1-py-
ridiniomethyl)-7,8-(D-a-sulfophenylacetamido)ceph-3-em-4-carboxylate monosodium salt] (Fig. 1) is a new semisynthetic cephalosporin which is selected from a series of 7-sulfophenyl-acetamido cephalosporins. It is a compound developed as a result of collaborative research between Takeda Chemical Industries, Ltd. and Ciba-Geigy Ltd. and was synthetized by Takeda Chemical Industries, Ltd. The relationships between chemical structures and microbiological activity of these cephalosporins were reported by Nomura et al. (11-13). The present report describes the in vitro and in vivo antibacterial activities of SCE-129 with carbenicillin as a reference compound. MATERIALS AND METHODS Antibiotics. SCE-129 was prepared by Takeda Chemical Industries, Ltd. Carbenicillin (Gripenin) was purchased from Fujisawa Pharmaceutical Co., and penicillin G (Penicillin G Kalium Takeda) was obtained from a commercial source. Bacteria. Laboratory strains were maintained on Trypticase soy agar (TSA) (BBL) or TSA supplemented with a 10% bovine blood slant (blood-TSA). Clinical isolates of Pseudomonas aeruginosa and Staphylococcus aureus were kindly supplied by S. Mitsuhashi, Gunma University and Y. Shimizu, the Central Clinical Laboratory; Osaka University Hospi-
tal, and were maintained on Dorset egg medium at
40C until use.
Determination of MIC. Minimum inhibitory concentration (MIC) was determined by the agar dilution method. One loopful (2 mm in diameter) of a suspension containing about 10' colony forming units (CFU)/ml or 106 CFU/ml, cultivated overnight on TSA, blood-TSA, or Tryticase soy broth (TSB) (BBL), was streaked for a length of about 2 cm on TSA, blood-TSA, or MacConkey agar (Eiken), or in TSB containing twofold serial dilutions of each antibiotic. The MIC was defined as the lowest concentration of antibiotic that prevented visible growth after overnight incubation at 37°C. Determination of MBC. Each tube of TSB containing twofold serial dilutions of each antibiotic was inoculated with test organisms to obtain a concentration of about 106 CFU/ml. After overnight incubation at 37°C, the MIC was obtained after inspecting the tubes for turbidity. A 2-pl portion (calibrated loop) from each tube was subcultured onto an antibioticfree TSA plate. The MBC was defined as the lowest concentration of antibiotic preventing visual growth on the subculture plate. Development of resistance in vitro. To generate resistance, the organisms were grown in 5 ml of TSB in the presence of twofold serial dilutions of each antibiotic. From the tube containing the highest concentration of the antibiotic that allowed normal or nearly normal growth, successive transfers were made every 48 h into the next series of tubes containing the same and higher concentrations. 137
138
TSUCHIYA, KONDO, AND NAGATOMO Hm-CO-NH
S_ 03.4o
o>'
2 3Co-"M2 42
FIG. 1. Chemical structure of SCE-129.
Bactericidal activity. Each TSB containing twofold serial dilutions of an antibiotic was inoculated with test organisms to obtain a concentration of about 106 CFU/ml. Control tubes contained no antibiotic. After incubation at 370C for 0, 2, 4, 6 and 8 h, portions from these cultures were plated on TSA. After 24 h of incubation, the number of colonies on each TSA plate was counted. Measurement of macromolecular substances in cells. S. aureus FDA 209 P was grown in TSB to the half-maximum growth. The cells were then harvested and divided into four portions: two were treated with SCE-129 to give concentrations of 1 and 10 tig/ml, respectively, one was treated with penicillin G to give a concentration of 0.05 Lg/ml, and the last one was used as a control. To monitor cell growth, the optical density of the bacterial suspension was measured at 650 nm by a Coleman Universal spectrophotometer. Portions of the culture were centrifuged, and the cells were washed twice with water. The protein fraction of each organism was isolated as described by Park male SLC-ddY mice weighing 19 to 23 g were infected of Lowry et al. (7). N-Acetylaminosugar was extracted and determined by the method of Reissig et al. (15). Nucleic acids from S. aureus were isolated according to the method of Schneider (17). Deoxyribonucleic acid was determined by the diphenylamine method and ribonucleic acid was determined by the orcinol method. Protective test. P. aeruginosa and S. aureus were cultured overnight in King A broth and brain heart infusion (Difco), respectively. The cultures were suspended in 5% mucin (Laboratories Division of Wilson Pharmaceutical and Chemical Co.). Four-week-old male SLC-ddY mice weighing 19 to 23 g were infected intraperitoneally with the organism suspended in 0.5 ml of 5% mucin. The challenge doses of P. aeruginosa and S. aureus were respectively about 100 times and 30 times the number of organisms required to kill 50% of the challenge control mice. Groups of five mice at each dose level were subcutaneously given 0.2 ml of antibiotics by following the schedule given in the table. All experiments were repeated four to five times. The 50% effective dose (ED50, mg/kg) was calculated by provit analysis from the survival rates recorded 7 days after infection (6).
ANTIMICROB. AGENTS CHEMOTHER.
aeruginosa and S. aureu& The antibacterial activities of SCE-129 and carbenicillin are illustrated in Fig. 2 and 3. Against 119 strains of P. aeruginosa, SCE-129 was about 10 times as active as carbenicillin. With the inoculum of 108 CFU/ml, 104 of 119 (87.4%) strains were inhibited at 1.56 to 25 utg of SCE-129 per ml, whereas only 1 of 119 (0.8%) strains was inhibited at 25 ,ug of carbenicillin per ml. Carbenicillin-resistant strains that were resistant to 400 jig of the penicillin per ml were susceptible to SCE-129 at 100 ug or less per ml. A similar pattern was observed on 106 CFU/ml as inoculum suspension. Against 112 strains of S. aureus, about 90% of which were resistant to penicillin G, SCE-129 showed activity similar to that of carbenicillin on iOW CFU/ml of inoculum suspension. With the inoculum of 101 CFU/ml, carbenicillin was more potent than SCE-129. Effects of inoculum size, medium pH, horse serum, and culture medium. The 50-SCE
4
-129
40
30-
0
m
II I
.
I
20
E
I
M
10
I114I /Il II
II/ I/
I
0.M 3C132.5
50gm200
4
I In I
0.
0.78 113 12.5
400
.50
200 . 400
MIC in pg/mi FIG. 2. Susceptibility of 119 clinically isolated strains of P. aeruginosa to SCE-129 and carbenicillin. MIC in' pg/ml
110
110
lOVmI
~~~~~~06.,,
100/m
100 90
90
-SCE-129 70
-70-
60
L_50
I
60
50o
40E40 RESULTS Z 30 oa 30 Antibacterial spectrum. SCE-129 was / found to be a unique antibiotic because the 20 o S S 20 4 ,/ 4 activity of SCE-129 was confined to P. aeruginosa and some gram-positive bacteria. It was 0 0 practically inactive against other gram-negative 0 39 1.56 6.22 25 0.39 1.56 6.25 25 rods. Various strains of P. aeruginosa showed MIC in IC in- pg/mi M pg/mi higher susceptibility to SCE-129 than to carbenicillin (Table 1). FIG. 3. Susceptibility of 112 clinically isolated Activity against clinical isolates of P. strains of S. aureus to SCE-129 and carbenicillin. I
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
VOL. 13, 1978
139
TABLE 1. Antibacterial spectrum of SCE-129 and carbenicillin MIC (ag/ml)
Mediumb
Organisma
SCE-129
Pseudomonas aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa
Escherichia coli
SP U 31 N 18 D 363 P8 NIHJ JC-1 Umezawa K-12 0-78 0-111 0-143 DT A
TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA TSA MCA MCA MCA MCA MCA
6.25 6.25 3.13 6.25 50 50 100 50 50 50 50 50 50 100 50 25 25 50 50 50 100 50 >100 >100 100 100 >100 100 >100 >100 100 3.13 6.25 6.25 3.13 1.56 3.13 3.13 25 25 6.25 6.25 1.56 100
Carbenicillin 50 100 3.13 100 100 12.5 6.25 25 6.25 1.56 3.13 6.25 50 6.25 12.5 6.25 6.25 12.5 3.13 12.5 12.5 3.13 3.13 1.56 6.25 1.56 50 1.56 6.25 0.78 3.13 0.39 0.78 12.5 0.2 0.2 0.2 0.2 1.56 0.39 0.2 0.2 1.56 0.39
E. coli E. coli E. coli E. coli E. coli Klebsiellapneumoniae Salmonella paratyphi S. schottmuelleri S. hirschfeldii S. typhi Boxhill-58 S. typhi Watson S. typhimurium Shigella dysenteriae EW-1 S. flexneri EW-10 S. flexneri EW-40 S. sonnei EW-33 Proteus mirabilis IFO 3849 P. mirabilis IFO 12255 P. vulgaris IFO 3851 P. vulgaris IFO 3988 P. vulgaris OX-19 P. vulgaris OX-K MCA P. morganii IFO 3168 MCA P. morganii IFO 3848 MCA Vibrio cholerae Inaba MCA FDA 209 P MCA Staphylococcus aureus S. aureus 308 A-1 MCA S. aureus 1840C MCA Streptococcus pyogenes E-14 Blood-TSA S. pyogenes Dick Blood-TSA S. pyogenes S-8 Blood-TSA S. pyogenes NY-5 Blood-TSA S. mitior America Blood-TSA S. pneumoniae type I Blood-TSA S. pneumoniae type II Blood-TSA S. pneumoniae type III Blood-TSA Corynebacterium diphtheriae Tronto Blood-TSA Bacillus subtilis PCI 219 Blood-TSA a Inoculum size: One loopful of bacterial suspension (10k CFU/ml). b TSA, Trypticase soy agar (BBL); Blood-TSA, TSA supplemented with 10% bovine blood; MCA, MacConkey agar (Eiken). c S. aureus 1840, Penicillin G-resistant strain.
MICs of SCE-129 against P. aeruginosa U 31 and D 363 and S. aureus FDA 209 P were determined under various conditions. The activity of SCE-129 increased as the inoculum size decreased. The MIC of SCE-129 was lower in an acidic pH than in an alkaline pH. The addition of 10 to 50% horse serum to the medium slightly increased the antibacterial activity of SCE-129. The activity of SCE-129 was not af-
fected by varying the culture medium (Table 2). Development of resistance in vitro. The resistance of P. aeruginosa U 31 to SCE-129 and carbenicilin was rapidly acquired. After the 4th transfer, the MICs of both antibiotics exceeded 1,600 ,ug/ml. Resistance of S. aureus to SCE-129 was acquired at a slightly slower rate than to carbenicillin. At the 23rd transfer, the
140
ANTimicRoB. AGENTS CHEMOTHER.
TSUCHIYA, KONDO, AND NAGATOMO
TABLE 2. Effect of various factors on the antibacterial activity of SCE-129 and carbenicillin MIC (jg/mi)
Carbeniciljin
SCE-129 Factor P. aeruginosa
Inoculum sizea 103 104 106 106 107 108 Medium pHb 6 7 8 9 Horse serum (%)C 0 10 20 50 Mediumd TSA NA MH HI BHI
S.
P. aerugmosa
aurew
aurew
U 31
D 363
0.78 1.56 1.56 3.13 3.13 6.25
25 50 50 50 100 200
25 25 25 50 50 100
0.05 0.1 0.2 0.2 0.39 0.78
1.56 3.13 3.13 6.25
3.13 3.13 6.25
6.25
100 100 200 200
25 50 100 100
0.39 0.39 0.78 0.78.
12.5 6.25 6.25 6.25
6.25 3.13 1.56 1.56
6.25 3.13 3.13 3.13
200 200 100 100
100 100 50 50
1.6 0.78 0.78 Q.78
6.25 6.25 6.25 6.25 6.25
3.13 3.13 3.13 3.13 6.25
U 31
D 363
1.56 3.13 3.13 6.25 6.25 12.5
0.78 1.56 1.56 1.56 1.56 3.13
6.25 6.25 12.5 12.5
FDA29P
.
FDAO9P
3.13 100 50 0.39 3.13 200 100 0.39 3.13 200 100 0.39 200 3.13 100 0.39 200 3.13 100 0.39 Inoculum site (CFU/ml): One loopful of bacterial suspension; Medium: TSA. b Inoculum sise: One loopful of bacterial suspension (106 CFU/ml); Medium: TSA. c Inoculum size: 0.1 ml of bacterial suspension (106 CFU/ml); Medium: TSB. d Inoculum size: One loopful of bacterial suspension (106 CFU/ml); Medium: TSA, Trypticase soy agar (BBL); NA, nutrient agar (Eiken); MH, Mueller-Hinton medium (Eiken); HI, heart infusion agar (Eiken); BHI, brain heart infusion agar (Eiken).
MIC of SCE-129 reached 400 itg/ml and, up to the 36th transfer, the MIC value did. not increase. The MIC of carbenicillin exceeded 400 ,ug/ml at the 16th transfer (Fig. 4). Cross resistance. The presence of cross resistance among SCE-129, carbenicillin, and four other cephalosporins was studied with highly resistant strains, which developed in vitro, of P. aeruginosa U 31 and S. aureus FDA 209 P. A cross resistance was observed between SCE-129 and carbenicillin in P. aeruginosa, and als among SCE-129, carbenicillin, and cephalexin in S. aureus, whereas the other three cephalosporins showed somewhat different patterns of cross resistance to S. aurezs (Table 3). Relationships between MIC and MBC. The MICs of SCE-129 determined by the. tube dilution assay closely resembled the MBCs against 9 strains of P. aeruginosa and 10 strains of S. aureus. The MIC and the MBC of SCE129 against P. aeruginosa were apparently lower than those of carbenicillin (Table 4). Bactericidal activity. SCE-129 was an effec-
S. aureus FDA 209 P
P. aeruginosa U 31
- - icdhl
1600
ICmb..icilli.
400-
I
100'
I
i400 -/ C
!
SCU-129
/
100
CE-12 6.25.
N
2
r
4e
Number of Transfer
I
i
10
is
20
25
,0 .5
Number of Transfer
FIG. 4. Patterns of development of resistance ofP. aeruginosa U 31 and S. aureus FDA 209 P to SCE129 and cqrbenicillin.
tive bactericidal agent against P. aeruguiosa and S. aureus. An apparent decrease in the number of living bacteria was observed at a concentration of 6.25 1g/ml for P. aerugnosa U 31 and 3.13 uAg/ml for S. aureus FDA 209- P, the concentrations being their MICs determiined by the tube dilution assay, and similar kiUl curves were observed at concentrations higher than 12.5 and 6.25 pg/ml, respectively (Fig. 5). Effect on the synthesis of macromolecu-
VOL. 13, 1978
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
141
TABLE 3. Cross resistance among SCE-129 and related antibiotics' MICb (Ag/Mi)
Organism
CB-PC
P. aeruginosa U 31 P. aeruginosa P. aeruginosa
(parent) r-CB-PC r-SCE-129
200 >1,600 >1,600
SCE-129
CER
CET
CEZ
CEX
25 200 >1,600
S. aureus FDA 209 P (parent) 0.39 3.13 0.05 0.1 0.2 1.56 S. aureus r-CB-PC >400 200 100 200 400 >400 S. aureus r-SCE-129 50 200 0.78 3.13 6.25 400 S. aureus r-CER 3.13 3.13 400 200 50 >400 S. aureus r-CET 25 12.5 100 400 400 >400 S. aureus r-CEZ 25 25 12.5 100 400 >400 S. aureus r-CEX 25 12.5 3.13 25 100 >400 a Inoculum size: One loopful of bacterial suspension (10' CFU/ml). Medium: TSA. b Antibiotics: CB-PC, carbenicdilin; CER, cephaloridine; CET, cephalothin; CEZ, cefazolin; CEX, cephalexin. TABLE 4. MIC and MBC of SCE-129 and carbenicillin for clinically isolated P. aeruginosa and S. aureus SCE-129 Carbenicillin MIC MIC MBC MBC Organism (pg/mi) (pg/mi) (jug/ni) (lg/mi) P. aeruginosa GN-3007-T 6.25 12.5 200 200 P. aeruginosa GN-3310-T 12.5 12.5 200 400 P. aeruginosa GN-3324-T 12.5 12.5 200 400 P. aeruginosa GN-3325-T 12.5 12.5 200 400 P. aeruginosa GN-3327-T 12.5 12.5 200 400 GN-3333-T 12.5 P. aeruginosa 12.5 200 400 GN-3338-T P. aeruginosa 12.5 12.5 400 400 GN-3363-T P. aeruginosa 6.25 6.25 50 100 P. aeruginosa GN-3365-T 6.25 6.25 100 100
S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus
T-3 T-15 T-20 T-30 T-32 T-62 T-63 T-69 T-78 T-88
6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.26 6.25
6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25
0.78 3 13 3.13 0.78 6.25 3.13 3.13 3.13 6.25 3.13
0.78 3.13 3.13 0.78 6.25 3.13 6.25 3.13 6.25 3.13
lar substances. The addition of 10 ,ug of SCE- cephalosporin administration. No protective ef129 or 0.05 ,ug of penicillin G per ml to a culture fect of SCE-129 was observed in a single 400of S. aureus resulted in growth suppression, but mg/kg subcutaneous dose 0, 1, 2, or 4 h after the growth of S. aureus was not affected by 1 infection (Table 5). As the frequency of the ,ug of SCE-129 per ml. The maxmum accumu- cephalosporin administration increased, the prolation of N-acetylaminosugar was found after 40 tective effect was enhanced. The protective efto 80 min of incubation with 10 ytg of SCE-129 fect was similarly potent in the groups treated or 0.05 ug of penicillin G per ml. The synthesis 0 and 2 h, 0 and 4 h, and 1 and 4 h after infection. of cellular RNA, DNA, and protein was not The effect in the triple dose was superior to significantly inhibited after 40 min of incubation that in the double doses; the dosage schedule with the indicated concentration of antibiotics of 0, 2, and 4 h after infection produced the most potent activity among the triple doses. (Fig. 6). Protective effects of various dosage Furthermore, four doses, given 0, 1, 2, and 4 h schedules. The effect of SCE-129 in mice in- after infection, showed the most potent protecfected with P. aeruginosa U 31 was markedly tive effect. From the above results, the further influenced by the frequency and interval of the evaluation of the protective effect of SCE-129
142
ANTIMICROB. AGENTS CHEMOTHER.
TSUCHIYA, KONDO, AND NAGATOMO
S. aureus FDA 209 P
P. aeruginosa U 31 10*
9. 8~
f
/
7.
o
jig /MI
0.78
pg/mI
1'.1.56 pg/ml .
cm
6-
C c
5
._
0 -
4,
3.13
Vk
6
,g/mi
25jp
jig/ml
3
:.>
2
K6 12.5ugm/ 25 jig/mi I
6
/m
i
i
Hours Hours U P. SCE-129 on aeruginosa 31 and S. aureus FDA 209 P. FIG. 5. Bactericidal effect of N-Ac.tyl-
Growth
1 .0
DNA
RNA
Protein
aminosugar
0.5S 0.3
0.6
0.6
0.2
0.4
0.4.
0.1
0.2
0.2
0.4-
a C a 0.6.
0.3.
U ._
0.4-
0.2-
0
0.2-
0.1.
4\
0.0.
0.0. 40
120
0.0*
0.0 40
120
40
120
40
120
40
120
Minutes FIG. 6. Effect of SCE-129 on growth, RNA, DNA, protein, and N. acetylaminosugar synthesis in S. aureus , SCE-129, 10 pg/ml; ------, 1 pg/mi; - - -, FDA 209 P. Symbols: -, Control; PCG, 0.05 pg/ml.
in P. aeruginosa-infected mice was performed by the dosage schedule 0, 2, and 4 h after infection.
Effect of challenge doses. The protective effect of SCE-129 in mice infected with various challenge doses of P. aeruginosa was studied (Table 6). P. aeruginosa NC-5 was about 100,000 tines more virulent than P. aeruginosa U 31. However, a similar protective effect was observed in mice infected with the miniimum number of organisms required to kill all untreated mice (1 MLD). In mice infected with P. aeruginosa NC-5 whose challenge dose was increased from 1 MLD (101 CFU/mouse) to 100 MLD
(103 CFU/mouse), the EDro values changed from 9.67 to 27.6 mg/kg, and further increase of the challenge dose produced a remarkable increase of ED5o values. The remarkable increase of ED5o values was observed in mice infected with a relatively low virulent strain, P. aeruginosa U 31. Comparative protective effects of SCE129 and carbenicillin. The protective effects of SCE-129 and carbenicillin were compared in mice infected with P. aeruginosa and S. aureus (Tables 7 and 8). P. aeruginosa N 18 was very susceptible to carbenicillin. Although MICs of both antibiotics against this strain were the
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
VOL. 13, 1978
TABLE 5. Effect of dosage schedule on ED50 of SCE129 against P. aeruginosa U 31 infection in mice2 Treatment (h) 0
1 2 4 0
+ 1
0+2 0+4 1+2 1+4 2+4
0+1+2 0+ 1+4 0+2+4 1+2+4
ED60 (mg/kg)b
>400 >400 >400 >400 >400 201 194 >400 243 >400
143
in mice SCE-129 was four times more potent than carbenicillin. In mice infected with P. aeruginosa D 363, SP, NC-5, and U 31, which show usual susceptibility against carbenicillin, SCE-129 was remarkably effective compared with carbenicillin. The protective effects of both same,
TABLE 6. Effect of challenge dose on ED60 of SCE129 against P. aeruginosa infection in mice4 ChalEDMm (mg/kg)b
(112-735) (153-258)
lenge
(173-424)
(CFU
j(al)
258 (178-546) 104 (81.2-134) 75.0 (58.8-99.5) >300
U 31 P. aeruginosa (MIC, 6.25 pg/ml)
NC-5 P. aerugnosa (MIC, 6.25 pg/ml)
101
9.67 (6.02-13.6) 19.9 (15.7-24.9) 27.6 (23.5-32.2) 48.4 (39.6-61.6) i05 207 (155-326) 10" 1,840 (1,320-3,410) 17.2 (13.8-21.8) 60.1 (49.5-77.5) >2,400 107 10 >2,400 >2,400 aMice were injected intraperitoneally with indicated CFU of test organism in 0.5 ml of 5% mucin. SCE-129 was administered subcutaneously 0, 2, and 4 h after infection. bED0 values were calculated as a total dose. Numbers in parenthess indicate 95% confidence limits.
102 103 104
0+1+2+4 51.0 (37.5-71.7) a MIC of SCE-129 against the organism was 6.25 jg/ml. Mice were injected intraperitoneally with 107 CFU of test organism per animal in 0.5 ml of 5% mucin. SCE-129 was administered subcutaneously. I ED80 values were calculated as a total dose. Numbers in parentheses indicate 95% confidence limits.
TABLE 7. Comparative protective effect of SCE-129 and carbenicillin on P. aeruginosa infection in mice4 P. aeruginosa
Antibioticb Antibiotic'
~
Challenge dose
(CFU/ainima) 106
MIC
Do(gk) D0(gk)
(gi
3.13 3.13
3.43 (2.80-4.13) 15.9 (13.2-19.2)
106
6.25 50
9.14 (7.08-11.4) 504 (408-615)
SCE-129 Carbenicillin
103
6.25 100
21.0 (17.5-25.4) 1,210 (1,040-1,420)
SCE-129 Carbencillin
107
6.25 100
60.1 (49.5-77.5) 1,720 (1,430-2,180)
N 18
SCE-129 CarbenicilHin
SP
SCE-129 Carbenicillin
NC-5 U 31
10" 438 (307-633) 6.25 SCE-129 100 >2,400 Carbenicillin a Mice were injected intraperitoneally with test organism in 0.5 ml of 5% mucin. b Antibiotics were administered subcutaneously 0, 2, and 4 h after infection. c ED50 values were calculated as a total dose. Numbers in parentheses indicate 95% confidence limits. D 363
TABLE 8. Comparative protective effect of SCE-129 and carbenicillin on S. aureus infection in micea AniitcChallenge MIC S. aureus Antibiotic EDro (mg/kg)b strain (g/niml) doC l 6.25 SCE-129 9.45 (7.16-12.5)" 10" 308 A-i
Carbenicillin
0.78 aMice were injected intraperitoneally with test organism in 0.5 ml of 5% mucin. b Antibiotics were administered subcutaneously 0 h after infection. c Numbers in parentheses indicate 95% confidence limits.
10.5 (8.13-13.8)
144
TSUCHIYA, KONDO, AND NAGATOMO
ANTIMICROB. AGENTS CHEMOTHER.
antibiotics in D 363-infected mice were weaker than those in mice infected with other strains, although MICs of D 363 to SCE-129 and carbenicillin, were similar in these strains. In vitro SCE-129 showed less activity against S. aureus 308 A-1 than did carbenicillin, but in mice SCE-129 was more potent than carbenicillin. Effect of administration route. SCE-129 was similarly effective in subcutaneous and intraperitoneal routes in mice infected with P. aeruginosa U 31. It is supposed that SCE-129 was well absorbed from the injection site. By the intravenous route the effect of SCE-129 was weaker. It may depend on a more rapid decrease of the plasma level. No protective effects were observed by the oral dose of cephalosporin (Table 9).
was not observed with SCE-129 at the concentration tested. It has been reported that cephalosporins, like penicillins, are inhibitors of cell-wall synthesis (1-5, 16). The accumulation of N-acetylaminosugar in the cells of S. aureus treated with SCE129 was observed at the concentration of 10 ,ug/ml, which did not affect the synthesis of DNA, RNA, and protein. It is suggested, therefore, that SCE-129 is an inhibitor of cell-wall synthesis of S. aureus. Bacteriolytic action of SCE-129 was observed with S. aureus but not with P. aeruginosa (unpublished data). Several cephalosporins showed bacteriolytic action against E. coli. Cephalosporin 7/30 [7-(S-benzylthioacetamido)-cephen-3-
DISCUSSION Against P. aeruginosa, SCE-129 was about 10 times more active than carbenicillin. The activity of SCE-129 against carbenicillin-resistant strains of P. aeruginosa was inferior to that against carbenicilhin-susceptible strains. The moderate activity of SCE-129 against carbenicillin-resistant P. aeruginosa may be attributed to the stability of SCE-129 against hydrolysis by /8-lactamase from carbencilin-resistant P. aeruginosa. The Vmax of SCE-129, which is expressed as the relative rate of hydrolysis, assuming that the rate for penicillin G was 100, was 0.2 for,B-lactamase from carbenicillin-susceptible strains of P. aeruginosa U 31 and 13 for ,Blactamase from carbenicillin-resistant strains of P. aeruginosa GN 3407. The Km of SCE-129 against the former ,B-lactamase was 192 mM, and the Km against the latterfl-lactamase was 7,100 mM (K. Okonogi et al., personal communication). Though several cephalosporins showed the so-called "paradox phenomenon" on bactericidal action against S. aureus (18), this phenomenon TABLE 9. Effect of administration route on ED50 of SCE-129 against P. aeruginosa infection in mice" Administration EDso (mg/kg)b route
65.1 (55.1-77.0) Subcutaneous ......... 59.6 (46.7-76.8) Intraperitoneal ......... Intravenous ......... 101 (80.8-129.8) Oral ......... >1,200 a Mice were injected intraperitoneally with 107 CFU of test organism per animal in 0.5 ml of 5% mucin. SCE-129 was administered 0, 2, and 4 h after infection. b ED5o values were calculated as a total dose. Numbers in parentheses indicate 95% confidence limits.
ylmethyl-N-dimethyldithiocarbamate-4-carboxylic acid)] inhibited the growth of E. coli, but did not reveal the bacteriolytic action (5). It is also reported that cephalosporin 7/30 inhibited the protein synthesis of E. coli (4). The mode of action of SCE-129 against P. aeruginosa needs further investigation. SCE-129 showed a potent protective effect in mice infected with P. aeruginosa. In vitro activity of SCE-129 was stronger than that of carbenicillin, and in vivo SCE-129 was frequently much more active than would be expected from its activity in vitro. For example, in vitro susceptibility of P. aeruginosa N 18 against SCE-129 was the same as that against carbenicillin, but in the protective test in N 18-infected mice, SCE-129 was four times more active than carbenicillin. In mice infected with P. aeruginosa SP or NC-5, SCE-129 was about 60 times more active, but only 16 times more active in vitro. In mice infected with S. aureus, SCE-129 was more active than carbenicillin, although in vitro activity of SCE-129 against this strain was less than that of carbenicillin. There are many reports concerning the effect of dosage schedule on the experimental murine infection. MacLeod and Stone (8) showed that the therapy by sulfonamide on pneumococcus type I infection in mice must be continued for a period of 48 to 72 h for recovery, but a considerable proportion of the animals survived by treatment with penicillin in triply divided doses at 4-h intervals on the day of infection. Zubrod (19) showed that in the treatment with multiple doses of penicillin G or penicillin K hemolytic streptococcus infection in mice, survival of the mice depends on the total dose administered over a fairly broad range of dosage schedules. Baker and Jackson (18) also reported that in streptococcal infection in mice, administering daily penicillin dosage in a series of small and equally spaced doses was not more effective than administering an equivalent amount in 1 or 2
SCE-129, ANTIPSEUDOMONAL CEPHALOSPORIN
VOL. 13, 1978
doses per day. In typhoid infection in mice, Miller et al. (9) showed that the double dosage of antibiotic in mice challenged with S. aureus and Salmonella schotmulleri was most effective and that by increasing the frequency of treatment, the total amount of antibiotic increased. The studies reported here indicate that in the treatment of acute intraperitoneal Pseudomonas infection in mice, the amount of SCE-129 necessary to protect varied by the dosage schedule. Little or no protective effect was observed after the doses at 0 and 1 h, 1 and 2 h, or 2 and 4 h postinfection compared with that of a single dose, but the protective effect of SCE-129 was observed on other double dosage schedules. A more potent effect was observed during the multiple dosage schedule. From the results indicated here, it seems that the administration at 0 h after infection is necessary to exert the potent effect. The effect of SCE-129 reflects the relationship between the cephalosporin concentration in the body fluid and the length of time this antibiotic sustained a high MIC value. The results with SCE-129 treatment in mice may not be directly transferrable to humans. Nevertheless, it is clear that these potent effects may be seen more frequently in multiple dosages under well controlled schedules rather than in a large single dose. ACKNOWLEDGMENT We are indebted to T. Iwahi and S. Shili for their technical asistance.
LITERATURE CITED 1. Bond, J. M., R. W. Brimblecomb, and R. C. Codner. 1962. Biological assay of cephalosporin C. J. Gen. Microbiol. 27:11-19. 2. Chang, T.-W., and L Weinstein. 1964. Morphological changes in Gram-negative bacilli exposed to cephalo. thin. J. Bacteriol. 88:1790-1797. 3. Chang, T.-W., and L Weinstein. 1964. Inhibition of synthesis of the cell wall of Staphylococcus aureus by cephalothin. Science 143:807-808. 4. Fountain, ILH., and A. D. Rusele. 1969. Studies on the mode of action of some cephalosporin derivatives. J. Appl. Bacteriol. 32:312-321. 5. Gonnella, J. S. 1967. In vitro and clinical effect of cephaloridine: a semisynthetic antibiotic. Am. J. Med. Sci.
145
254:93-104. 6. Litchffeld, J. T., and F. Wilcoxon. 1949. A simple method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96:99-113. 7. Lowry, O. H., J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 8. MacLead, C. H., and E. R. Stone. 1945. Differences in the nature of antibacterial action of the sulfonamides and penicillin and their relation to therapy. Bull. N.Y. Acad. Med. 21:375-388. 9. Miller, A. K., D. L Wilmer, and W. F. Verwey. 1950. Effect of penicillin dosage schedule on treatment of experimental typhoid in mice. Proc. Soc. Exp. Biol. Med. 74:62-64. 10. Miller, A. K, and H. Kropp. 1967. Antibacterial chemotherapy tests. II. Influence of route and number of treatments on in vivo effectiveness of streptomycin and tetracycline and influence of resistance to rechallenge, p. 377-381. Antimicrob. Agents Chemother. 1966. 11. Nomura, H., T. Fugono, T. Hitaka, I. Minami, T. Azuma, S. Morimoto, and T. Masuda. 1974. Semisynthetic ,8-lactam antibiotics. 6. Sulfocephalosporins and their antipseudomonal activities. J. Med. Chem. 17:1321-1315. -12. Nomura, H., T. Fugono, T. Hitaka, I. Minami, T. Azwna, S. Morimoto, and T. Masuda. 1974. Semisynthetic ,44actam antibiotics. VII. New semisynthetic cephalosporinsr derived from a-sulfophenylacetic acid. Heterocycles 2:67-72. 13. Nomura, H., I. Minami, T. Hitaka, and T. Fugono. 1976. Semisynthetic ,B-lactam antibiotics. 8. Structureactivity relationships of a-sulfocephalosporins, J. Antibiot. 29:928-936. 14. Park, J. T., and R. Hancock. 1960. A fraction procedure for studies of the synthesis of cell-wall mucopeptide and of other polymers in cells of Staphylococcus aureus. J. Gen. Microbiol. 22:249-258. 15. Reisig, J, L, J. L Stromiuger, and L F. Leloir. 1955. A modified colorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217:959-966. 16. Russell, A. D,, and R. H. Fountain. 1971. Aspects of the mechanism of action of some cephalosporins. J. Bacteriol. 106:65-69. 17. Schneider, W. C. 1957. Determination of nucleic acid in tissues by pentose analysis. Methods Enzymol. 3:680-684. 18. Tsuchiya, K., T. Nishi, T. Oishi, S. Goto, and Y. Kaneko. 1976. Antibacterial activity of cephacetrile. Chemotherapy (Tokyo) 24:1-15. 19. White, E. J., M. J. Baker, and E. R. Jackson. 1948. Therapeutic effectiveness of single and divided dose of penicillin in a streptococcal infection in mice. Proc. Soc. Exp. BioL Med. 67:199-203. 20. Zubrod, C. G. 1947. Comparative efficiency of single and multiple dosage regiments of the penicillins. Bull. Johns Hopkins Hosp. 81:400-410.
