ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1975, p. 306-310 Copyright © 1975 American Society for Microbiology

Vol. 7, No. 3 Printed in U.S.A.

BL-S640, a Cephalosporin with a Broad Spectrum of Antibacterial Activity: Bioavailability and Therapeutic Properties in Rodents F. LEITNER,* D. R. CHISHOLM, Y. H. TSAI, G. E. WRIGHT, R. G. DEREGIS, AND K. E. PRICE Department of Microbiological Research, Bristol Laboratories, Syracuse, New York 13201

Received for publication 30 December 1974

The bioavailability and therapeutic properties of BL-S 640 in rodents were compared with those of cephalothin, cephaloridine, and cefazolin after parenteral administration, and cephalexin after oral administration. When given intramuscularly in dosages of 5 to 40 mg/kg, peak concentrations of BL-S 640 in the blood of mice were proportional to dose, but when given orally, they were proportional only up to a dose of 25 mg/kg. After either route of administration, the concentration of BL-S 640 in the blood declined at a slower rate than that of the control compounds. Rats receiving BL-S 640 orally excreted an average of 39% of the drug in the urine. BL-S 640 was highly effective in the treatment of mice infected systemically with a variety of pathogenic bacteria, its therapeutic efficacy in comparison with that of other cephalosporins being frequently in excess of what would have been predicted on the basis of comparative activities in vitro. The bioavailability and therapeutic properties of BL-S 640 were assessed by determining its concentration in blood, the percentage of recovery in urine, and the efficacy of the drug in the treatment of experimental infections after parenteral or oral administration to rodents. In studies using the parenteral route of administration, BL-S 640 was compared with cephalothin, cephaloridine, and cefazolin; in those involving the oral route, cephalexin was the

control compound. MATERIALS AND METHODS Cephalosporins and bacteria. The antimicrobial agents and microorganisms used in this study have been described previously (1). Antibiotic concentration in blood. Male SwissWebster mice, weighing 20 (±1) g, were given 0.2 ml of antibiotic solutions at appropriate concentrations either by intramuscular injection or by gavage. The vehicle for the parenteral route was 0.01% phosphate buffer at pH 7.0, and for the oral route Tween-CMC (a preparation containing 0.93% Tween 40 and 0.4% carboxymethylcellulose HV Type 70). Eight animals were used for each dose level. Those receiving the compound per os were fasted for 4 h prior to drug administration. Blood samples (0.03 ml) were obtained from the orbital sinuses by means of heparinized capillary tubes (Clay Adams) at 0.25, 0.5, 1, and 1.5 h after intramuscular (i.m.) and 0.5, 1, 2, and 3.5 h after oral administration of the compound. Paper disks, 6.35 mm in diameter, were impregnated with the blood, and the antibiotic activity was assayed by the diffusion technique using seed agar (BBL) inoculated 306

with Bacillus subtilis ATCC 6633 or Sarcina lutea ATCC 9497. A standard line relating the diameter of the inhibition zone to drug concentration was obtained by assaying the compounds at known concentrations in heparinized mouse blood. Recovery in urine. Male Sprague-Dawley rats, weighing 200 (±10) g, received a dose of 50 mg of cephalosporin per kg in 5 ml of Tween-CMC by gavage. Four rats were used per compound. The animals were fasted for 18 h before dosing. Although water was available ad libitum, the animals were hydrated with 5 ml of water at 3 and 6 h after dosing. The rats were housed individually in metabolism cages, and urine specimens were collected over dry ice during intervals of 0 to 6 and 6 to 24 h after drug administration. Portions (0.03 ml) of appropriate dilutions of urine were placed on paper disks (6.35 mm in diameter), and the antibiotic activity was assayed by the diffusion technique on seed agar inoculated with B. subtilis ATCC 6633. A standard line relating the diameter of the inhibition zone to drug concentration was obtained by assaying the compounds at known concentrations in urine collected from untreated control animals. Treatment of systemically infected mice. Male Swiss-Webster mice, weighing 20 (41) g, were challenged intraperitoneally with 0.5 ml of a bacterial suspension containing sufficient organisms to kill untreated animals within 72 h. The suspending medium for Streptococcus pneumoniae, Streptococcus pyogenes, and Klebsiella pneumoniae was brain heart infusion broth (Difco). Staphylococcus aureus no. 2 was suspended in broth containing 2% mucin; the remaining organisms were suspended in medium containing 4% hog gastric mucin (type 1701w, Wilson Laboratories, Inc, Park Forest South, Ill.). The ani-

IN VIVO PROPERTIES OF BL-S 640

VOL. 7, 1975

mals received 0.2 ml of appropriate concentrations of antibiotic either by i.m. injection or by gavage. Five mice were used for each dose level. The animals were treated twice in accordance with a schedule given in the tables. The number of mice surviving the challenge for 3 or 5 days (depending on the test organism) was recorded and the PD50-the dose in mg/kg required to protect half the infected animals from death-was then estimated by means of a log probit plot. Treatment of intracystically infected rats. The bladder of female Long-Evans rats, weighing approximately 225 g, was surgically exposed under anesthesia. Urine was drained, and a challenge of 1.8 x 107 cells of Proteus mirabilis strain no. 1 was injected into the bladder. After positioning the bladder in place the incision was closed with wound clips. Treatment with BL-S 640 or cephalexin was initiated 24 h after infection. The following doses were given orally, four times a day for 3 days, to groups of eight animals per dose level: 0.04, 0.2, 1, 5, and 25 mg/kg. Ten animals were challenged but left untreated, and five animals were sham operated. The rats were autopsied four days after being infected, and the number of viable organisms in the bladder and the kidneys was determined.

307

TABLE 1. Relative area under the blood level curve of micea f Cdt/f C,rfdt Dose Cepha- Cepha- Cefa- BL-S CephaRot' mg/kg ute _____ lothin loridine zolin 640 lexin i.m. 5 1.0 1.2 2.1 3.3 10 1.0 1.9 3.0 4.2 20 1.0 2.1 3.2 4.2 40 1.0 1.9 3.0 4.0

P.O.

3.1 6.3 12.5 25 50 100

3.5 3.3 2.7 2.1 2.0 1.8

1.0 1.0 1.0 1.0 1.0 1.0

aReference compounds were cephalothin for intramuscular and cephalexin for oral administration. Limits of integration were 0 to 1.5 h for the intramuscular and 0 to 3.5 h for the oral route. b i.m., Intramuscular; p.o., per os.

RESULTS Bioavailability studies. (i) Concentration in murine blood. When given i.m., peak antibiotic concentrations of BL-S 640 and the three control compounds were observed 0.25 h after drug administration. This is illustrated in Fig. 1 (left panel) for a dose of 20 mg/kg. At this dose, cefazolin reached a peak concentration of TIME (h) DOSE (rm/kg) 34 gg/ml, BL-S 640 of 32 ,ug/ml, cephaloridine FIG. 2. Antibiotic concentration in blood after oral of 23 gg/ml, and cephalothin of 17 ,g/ml. The concentration of BL-S 640 in the blood declined administration to mice. Symbols: 0, BL-S 640; 0, at a slower rate than that of the control cepha- cephalexin. losporins. With all compounds peak concentraAreas (fCdt) under the blood level curves (C), tions were proportional to dose in the range for 0 < t < 1.5 h, were determined and related to tested (Fig. 1, right panel). cephalothin, chosen as the reference compound. Relative areas (JCdt/fCre,dt) for cephaloridine, cefazolin, and BL-S 640 increased between the doses of 5 and 10 mg/kg and remained virtually O. 25 h TME: ,a constant for doses of 10, 20, and 40 mg/kg 30 60Q (Table 1). At these higher doses, the relative /O area was 2 for cephaloridine, 3 for cefazolin, and X ... E

z

u

20

40
400

7

24

36

50

6 7

2 x 10'-4 x 106 2 x 104-5 x 104

51

> 400

> 400

> 400

15

>400

>400

>400

1 6

0.7 x 107-2 x 107

0.7

55

20

22

7

3 x 104

0.8 1

22 31

7 7

11

1

0.2 x 10'-2 x 106 0.8 x 105-7 x 105

0.3

12

7

7

0.7

47

6

15

3

2 x10

2x

10'-5 x 10

10

9

2 x 104-4 x 104 0.6 x 10'-3 x 106

30

> 400

180

90

2

82

>400

140

200

Proteus rettgeri

3

4 x 106-6 x 106

22

92

27

23

Providencia stuartii

1

4

145

67

12

Providencia alcalifaciens

1

10'-2 x 105 2 x 106-5 x 10'

9

150

36

13

Proteus morganii

a Animals

1

1x

were treated twice: 1 and 3.5 h after infection.

VOL. 7, 1975

IN VIVO PROPERTIES OF BL-S 640

309

TABLE 3. Efficacy of the intramuscular treatment of mice systemically infected with gram-positive organismsa

PD50 per treatment (mg/kg) Organism

Challenge (no. of organisms)

Strain

Staphylococcus aureus

BL-S 640

Cephalothin

loridine

Geaoi

Cepha-

Cefazolin

1 2 3

2 x 105 1 x 109-4 x 109 3x108

0.07 6

0.6 6

0.05 4

0.4 9

2

6

1

2

Streptococcus pneumoniae

1 2

1 X 104 1 X 105-2 x 105

0.7 0.6

0.15 0.6

0.5

Streptococcus pyogenes

1 2

3 x 104 0.2 x 105-1 X 105

0.15 0.03

15 66 6 1.3

0.08 0.02

0.9

2 0.3

[

a Animals infected with penicillinase-producing staphylococcal strains (no. 2 and 3) were treated 0 and 2 h after infection, the others were treated 1 and 3.5 h after infection.

TABLE 4. Efficacy of the oral treatment of mice systemically infected with gram-negative organismsa Organism

|

Escherichia coli

Strain

|

Challenge (no. of organisms)

(mg/kg) PD50 per treatmentCephalexin BL-S 640

1 2

2 x 105-3 x 105 0.7 x 105-2 x 105

1 7

2 x 104-3 x 104 2 x 103

1 16

47 >400

Enterobactercloacae

7

3 x 104-4 x 104

8

110

Proteus mirabilis

1 6 7

0.1 x 107-1 x 107 2 x 106-5 x 106 2 x 104-3 x 104

1.3 1.1 0.5

49 30 40

Proteus vulgaris

1 3

0.2 x 101-2 x 106 0.1 X 101-1 X 106

1.1 1.1

36 36

Proteus morganii

1

1 x 104-2 x 104

50

>400

Providencia stuartii

1

0.1 x 101-2 x 106

6

48

Providencia alcalifaciens

1

2 x 101-5 x 10

14

120

Klebsiella pneumoniae

a

Animals were treated twice:

1.2 2

21 14

1 and 3.5 h after infection.

TABLE 5. Efficacy of the oral treatment of mice systemically infected with gram-positive organismsa Organism

Staphylococcus aureus

Strain

1

2 3

Streptococcus pneumoniae

Streptococcus pyogenes

Challenge (no. of organisms) 2 x 105-3 x 105 1 x 109-4 x 109 1 X 108-4 x 108

(mg/kg) PD,0 per treatmentCephalexin

BL-S 640

0.09 26 9

33 6

0.15

0.2 x 104-2 x 104 6 x 104-8 x 104

0.6 0.4

30

2 1

0.9 x 104-2 x 104 0.7 x 106-1 X 106

0.14 0.08

3

1

2

a Animals infected with penicillinase-producing staphylococcal strains (no. 2 and 3) after infection, the others were treated 1 and 3.5 h after infection.

37 3 were

treated 0 and 2 h

ifto

LEITNER ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 6. Efficacy of the oral treatment of intracystically infected ratsa Log,0 of viable organisms Bladder

Dose (mg/kg)

25 5 1 0.2 0.04

Kidneys

BL-S 640

Cephalexin

BL-S 640

Cephalexin

2.0 0.9 2.3 4.2 7.4

1.4 2.8 6.9 6.8 6.4

0.8 1.8 2.8 2.5 6.7

1.1 1.8 5.0 6.2 6.0

a The animals were treated 4 times a day for 3 days with the indicated doses. The infecting organism was Proteus mirabilis strain no. 1; the challenge was 1.8 x 107 organisms. Cell counts were performed 4 days after challenge. Log1o of viable organisms with zero dose was 7.4 in the bladder and 7.5 in the kidneys.

reduce the number of infecting organisms in the bladder and the kidneys in comparison with animals left untreated. Four days after infection with a strain of P. mirabilis, these organs contained about 25 to 30 million viable organisms in untreated rats, which is virtually identical with the challenge (Table 6). BL-S 640 reduced the number of infecting organisms to less that 1,000 in the bladder when 1 mg/kg, and in the kidneys when 0.2 mg/kg, were administered four times a day for 3 days. A dose of 5 mg/kg was required to achieve comparable results by the same regimen with cephalexin. DISCUSSION Compared to the other cephalosporins, the therapeutic efficacy of BL-S 640 was frequently in excess of what would have been predicted on

the basis of of its comparative activity in vitro (1). A few examples will illustrate this point. BL-S 640 was about 10 times more effective than cefazolin against infections with two strains of E. coli (Table 2) but only half as active as cefazolin against the same strains in vitro. Similarly, BL-S 640 and cephaloridine were about equally effective in the treatment of animals infected with strains 1 and 2 of S. aureus (Table 3), although in vitro cephaloridine was 64 times more active than BL-S 640. Compared with cephalexin against two strains of S. pneumoniae, BL-S 640 was 50 to 90 times more effective in vivo (Table 5) but only 16 times more active in vitro. It is not unreasonable to assume that the considerably longer half-life of BL-S 640 in blood contributed to its therapeutic superiority. In view of its antibiotic and therapeutic properties, we believe that BL-S 640 deserves further consideration as an antibiotic agent for clinical use. ACKNOWLEDGMENTS We are indebted to Celeste C. Carmack, Violet S. Mallor, Antoinette C. Nutting, Erwin B. Williams, and Ellen M. Yastrib for devoted technical assistance. We acknowledge the excellent clerical assistance of Evelyn Barenholtz and Maxine C. Postle.

Addendum in Proof Cefatrizine is the non-proprietary name of BL-S 640, recently approved by the USAN Council. LITERATURE CITED 1. Leitner, F., R. E. Buck, M. Misiek, T. A. Pursiano, and K. E. Price. 1975. BL-S 640, a cephalosporin with a broad spectrum of antibacterial activity: properties in vitro. Antimicrob. Agents Chemother. 7:298-305.

BL-S640, a cephalosporin with a broad spectrum of antibacterial activity: bioavailability and therapeutic properties in rodents.

The bioavailability and therapeutic properties of BL-S 640 in rodents were compared with those of cephalothin, cephaloridine, and cefazolin after pare...
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