Vol. 11, No. 1 Printed in U.S.A.

ANTIMICROBIAL AGENTS AND CHEICOTHERAPY, Jan. 1977, p. 51-63 Copyright © 1977 American Society for Microbiology

Laboratory Evaluation of FR10024, Derivative

a

New Cephalosporin

MINORU NISHIDA, TAKEO MURAKAWA, TOSHIAKI KAMIMURA, NAOHIKO OKADA, SHIGEMI FUKADA, HIROSHI SAKAMOTO, SHOJI NAKAMOTO, YOSHIKO YOKOTA, AND YOKO KONO Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan

Received for publication 20 July 1976

FR10024 is a broad-spectrum antibiotic. The in vitro antibacterial activity of FR10024 against clinical isolates of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis is greater than that of any of the cephalosporins developed to date. Indole-positive Proteus, Enterobacter, and Citrobacter are resistant to FR10024, as is true for the other cephalosporins. However, more than half of the strains of Enterobacter and Citrobacter tested were susceptible to FR10024 at an inoculum of 106 cells/ml. A single subcutaneous injection of FR10024 to mice with peritoneal infections due to S. aureus and several species of gram-negative bacilli gave a protective effect inferior to that of cefazolin but appeared to be superior to that of cephalothin. When given in two divided doses, however, the protective effect of FR10024 was enhanced and almost equaled that of cefazolin. The serum levels and rates of urinary recovery of FR10024 varied in different animal species. The mean peak serum level of FR10024 in humans after a single intramuscular injection of 500 mg was two times higher than that of cephalothin. The serum half-life after intramuscular injections of 250 and 500 mg was slightly shorter than that of cephalothin. After receiving 250 mg of FR10024 intramuscularly the urinary recovery rate was 87.7% in healthy volunteers. The biliary excretion rate of FR10024 was particularly high. The 24-h excretion of FR10024 in rats was 63.3%, this being six to seven times higher than that for cefazolin, which has the highest biliary excretion of the other known cephalosporins. When FR10024 was injected intramuscularly (20 mg/kg), it was found that the hepatic levels of FR10024 in rats were the highest of all the cephalosporins, including cefazolin, but the levels of FR10024 in other tissues were not as high as those of cefazolin. cefazolin by Fujisawa Pharmaceutical Co., Ltd.; cephaloridine, cephalothin, and cephalexin by Eli Lilly & Co.; cephapirin by Bristol Laboratories; and cephacetrile by Ciba Geigy AG. Subjects. The antibiotics were tested in the following subjects: (i) male ICR strain mice aged 4 weeks, each weighing 21 to 25 g; (ii) male SD strain rats aged 6 weeks, each weighing 190 to 240 g; (iii) male beagle dogs, each weighing 9.5 to 14.5 kg; (iv) male rhesus monkeys, each weighing 6.4 to 9.9 kg; (v) male healthy adult volunteers, each weighing 56 to 65 kg. In vitro antibacterial activity. The in vitro antibacterial activity of the test antibiotics was determined by the agar dilution method (2). One drop of an overnight broth culture and decimal dilutions thereof were inoculated on heart infusion agar (Difco) containing graded concentrations of the test drugs. The minimum inhibitory concentration (MIC) was determined after incubation at 37°C for 20 h. Bactericidal activity. (i) MBC. Each heart infusion broth containing graded concentrations of the test antibiotics was inoculated with test organisms to obtain a concentration of about 106 cells/ml. After

In an effort to develop better cephalosporins, great number of derivatives have been synthesized and evaluated in the Research Laboraa

tories of Fujisawa Pharmaceutical Co., Ltd., Osaka. Cefazolin (4) and ceftezole (5), both having excellent antibacterial activity and tolerance, were found in this screening program and both are now in clinical use. FR10024, a new cephalosporin derivative [sodium 7-(2-thienyl)acetamido-3- (1-methyltetrazol-5-yl)-thiomethyl-3-cephem-4-carboxylate] recently developed in our Research Laboratories, has proved superior in antibacterial activity to both cefazolin and ceftezole (Fig. 1). In animal experiments with FR10024, it was found that its biliary excretion pattern was unqiue when compared with that of other known cephalosporins (1, 3-5). We report here the results obtained thus far. MATERIALS AND METHODS Test antibiotics. FR10024 and cephalosporin C were provided by Fujisawa Research Laboratories; 51

52

ANTIMICROB. AGENTs CHEMOTHER.

NISHIDA ET AL. I

cic~~~

QC

Io NIN

H2

COI HHAL COONa

1

,.-N

CH3

!

FR10024

R1

CEPHALOTHIN

I

CH CO-

-

OCOCH3 N-N

CEFAZOLIN

-j_CH2CO-

CEFTEZOLE

NCH2CO_

S

CH3

_t

FIG. 1. Chemical structure of FR10024 and other cephalosporins. incubating at 37°0 for 20 h, the minimum bactericidal concentration (MBC) was determined as the lowest antibiotic concentration in which viable cell counts were decreased to less than 102 cells/ml. (ii) Initial killing rate. Heart infusion broths containing a concentration of 10 ,ug of each of the test antibiotics per ml, were inoculated with four strains of Escherichia coli or Klebsiella pneumoniae (106

cells/ml) and were then incubated at 370C for 60 min. After inactivation with Citrobacter 3-lactamase, which destroyed the residual antibiotic in 5 min at 370C, viable cell counts in the culture fluids were measured. Serum-protein binding. A 0.5-ml portion of the antibiotic solution (300 i,g/ml), in 0.067 M phosphate buffer (pH 7.0), was added to 4.5 ml of fresh serum and was then incubated at 370C for 1 h. This reaction liquid was poured into a Visking tube (size 8/32), suspended in a 15-ml polypropylene tube, and centrifuged at 1,000 x g for 30 min (10). The free antibiotic concentration in the ultrafiltrate was determined by the disk method (2). Protective effect on infections in mice. Staphylococcus aureus, E. coli, K. pneumoniae, Proteus mirabilis, and Citrobacter freundii, precultured overnight on agar slants at 370C, were suspended in a mucin solution to obtain specified cell counts of test organisms. Male ICR strain mice aged 4 weeks, each weighing 21 to 25 g, were used, and each group consisted of 10 mice. Mice were inoculated intraperitoneally with 0.5 ml of the suspension and were given a single subcutaneous dose of the test antibiotics 1 h after challenge. In another experiment, mice were given two divided doses of the test antibiotics 1 h and either 2 or 3 h after challenge. The 50% effective dose values were found by the probit method from the number of mice surviving after 2 weeks of observation.

Serum levels. The test antibiotics were injected intramuscularly (i.m.) in animals (20 mg/kg) and in human volunteers (250 and 500 mg). The serial data on rats were obtained on the different groups used at each bleeding, and the serial data in other animals and volunteers were obtained on the same subjects throughout the test period. The antibiotic levels in each serum sample were determined by the disk method, using standard solutions prepared with animal and human sera. The serum half-life was calculated from the linear descending slope of mean antibiotic concentrations in the serum plotted se,milogarithmically against time. Urinary excretion. Urine of rats, monkeys, and healthy volunteers was collected at specified intervals (5) after i.m. injection of the test antibiotics, 20 mg/kg in rats and monkeys and 250 and 500 mg in healthy volunteers. The antibiotic levels in each urine sample were bioassayed by the standard solutions prepared with 0.067 M phosphate buffer, pH 7.0, because there were no significant differences in standard curves between the standard solutions prepared with the buffer and those prepared with urine. Biliary excretion. Rats and dogs anesthetized intraperitoneally with pentobarbital (25 mg/kg) were fixed in the supine position, and a polyethylene cannula was inserted into the bile duct. Bile samples were collected at 0 to 3, 3 to 6, and 6 to 24 h after i.m. injection of 20 mg of the test drugs per kg. The antibiotic levels in the bile samples were bioassayed with the standard solutions prepared with 0.067 M phosphate buffer, pH 7.0, for the same reason as in the case of urine. Identification of active antibiotic substances in urine and bile samples. After i.m. injection of FR10024, urine samples were collected from rats, monkeys, and healthy volunteers, and bile samples were collected from rats at specified intervals. Each sample was examined by thin-layer chromatography and bioautography. For thin-layer chromatography, the following solvent was used: n-butanolacetic acid-water (4:1:2). The absorbent was Eastman chromagram sheet no. 6061. For bioautography, sheets developed with the above solvent were dried and set on the agar media, which were inoculated with 0.2% spore suspensions of Bacillus subtilis ATCC 6633 (2 x 108 spores/ml) for approximately 15 min. After the sheets were removed and the agar plate was incubated at 370C for 20 h, the bioactive substances were identified. Tissue distribution. Rats that had received 20 mg of the test antibiotics per kg were bled at set intervals and their organs were removed. After the organs were lightly washed with saline solution, the tissues were homogenized in twice their weight of ethanol with the polytron homogenizer. The antibiotic levels in the supernatant, obtained by centrifuging the homogenate at 10,000 x g for 10 min, were bioassayed with a standard solution prepared with 0.067 M phosphate buffer at pH 7.0 containing 66% ethanol, since there were no significant differences in standard curves between the standard solution with 0.067 M phosphate buffer, pH 7.0, containing 66% ethanol and the supernatant obtained from the control tissues in the same manner. The tissue levels of the test drugs were thus calculated.

53

LABORATORY EVALUATION OF FR10024

VOL. 11, 1977

RESULTS

Antibacterial activity: Antibacterial

spec-

trum. FR10024, like other well-known cephalo-

sporins, is a broad-spectrum antibiotic active against gram-positive and gram-negative organisms. When it was compared with other antibiotics for antibacterial activity against various standard strains, FR10024 was slightly more active against gram-positive bacteria than cefazolin and cephacetrile but less active than cephaloridine. However, the activity of FR10024 against gram-negative bacteria was greater

than that of any other cephalosporin tested. FR10024 was less active against Streptococcus faecalis 6733 and was inactive against Proteus vulgaris IAM-1025 and Pseudomonas aeruginosa IAM-1095 (Table 1). Susceptibility distribution. MICs ofFR10024 against 42 strains of S. aureus ranged from 0.05 to 0.39 ,ug/ml for an inoculum of 106 cells/ml, and no strains resistant to FR10024 were found (Table 2). The antibacterial activity of FR10024 against S. aureus was greater than that of cefazolin. The MICs of FR10024 against 21 strains ofS.

TABLE 1. Antibacterial spectra ofFR10024 and other cephalosporinsa

(pg/ml)

MIC

Organism

FR10024

S. aureus 209P JC-1 S. aureus Newman S. aureus Smith B. subtilis ATCC 6633 M. luteus PCI-1001 S. pneumoniae IIIb S. pyogenes S-23b S. faecalis 6733b C. diphtheriae PW-8b

Cefazolin Cephaloridine

0.39 0.78 0.39 0.39 0.39 0.39 0.2 50 0.1

0.1 0.2 0.1

0.025 0.05 0.2 0.1 25 0.05

0.1 0.1 0.025 0.05

0.025 0.1 0.05 25 0.05

Cephalothin

Cephapirin

0.39 0.39 0.2 0.05 0.1 0.2 0.1 25 1.56

Cephace-

trie

0.39 0.78 0.39 0.2 0.39 0.2 0.39 12.5 0.2

0.1

0.1 0.1 0.025 0.05 0.2 0.05 25 0.05

12.5 12.5 >100 >100 6.25 12.5 6.25 12.5 12.5 12.5 12.5 6.25

50 6.25 >100 >100 3.13 3.13 6.25 6.25 3.13 12.5 25 12.5

6.25 25 3.13 3.13 E. coli NIHJ JC-2 12.5 25 3.13 1.56 K. pneumoniae NCTC 418 100 100 P. vulgaris IAM-1025 100 >100 >100 >100 >100 >100 P. aeruginosa IAM-1095 3.13 1.56 1.56 S. typhi T-287 0.2 3.13 3.13 S. paratyphi A 1015 0.1 3.13 3.13 12.5 S. schottmuelleri 8006 0.2 3.13 3.13 3.13 S. typhimurium 1406 0.2 3.13 3.13 3.13 S. enteritidis 1891 0.2 3.13 6.25 12.5 S. dysenteriae Al Shiga 0.78 3.13 6.25 6.25 6.25 S. flexneri la EW-8 0.39 1.56 6.25 S. sonnei I EW-33 0.78 0.78 a Conditions: Heart infusion streak method, 370C, 20 h, 108 cells/ml. b Supplemented with 10% rabbit blood.

TABLE 2. Distribution of susceptibilities of clinical isolates to FR10024 and other cephalosporinsa No. of strains showing MIC (,ug/ml) of: Antibiotic Organism

S. aureus (42 strains)

0.05

0.1

0.2

0.39

0.78

1.56

FR10024 Cefazolin

1

4

10 23

16

1

Cephaloridine Cephalothin

7

29 1 1

27 2 6 18 25

Cephapirin Cephacetrile S. faecalis (21 strains)

a

22 16 1

3.13

6.25

12.5

25

50

11 13

15

10 8 5

18 1

14

3 20 6

1

15

24

2

FR10024 Cefazolin

Cephaloridine Cephalothin Cephapirin Cephacetrile Conditions: Heart infusion agar, stamp method, 37°C, 20 h, 106 cells/ml.

.1

1

100

54

ANTIMICROB. AGENTS CHEMOTHER.

NISHIDA ET AL.

strains ranged from 12.5 to 50 ,ug/ml. No highly resistant strains were observed. FR10024 had the best antibacterial activity of all the cepha-

faecalis ranged from 25 to 50 ,ug/ml. The activity of FR10024 was almost the same as that of cefazolin and was weaker than that of cephaloridine, cephapirin, and cephacetrile. Table 3 shows the distribution of susceptibility of each of the 42 strains of E. coli, K. pneumoniae, andP. mirabilis to the antibiotics. For E. coli, the MICs of FR10024 ranged from

SO6Qa :e .'a a) a)a

covery in rats was due to the high biliary excretion of FR10024, as mentioned below. (ii) Monkeys. The 24-h urinary recovery rate of FR10024 in monkeys after i.m. injection of 20 mg/kg was compared with that of the other test drugs (Table 11). The recovery rate of FR10024 in monkeys, unlike that in rats, was 76.6%, i.e., almost the same as that of cefazolin (80.3%) and cephaloridine (74.4%). However, the recovery rate of FR10024 varied with the species, as was the case for serum levels. (iii) Healthy volunteers. The 24-h urinary recovery rate of FR10024 in five healthy volunteers after i.m. injection was as high as 87.7% for 250 mg and as high as 88.5% for 500 mg (Table 12). The peak urinary levels of FR10024 after administration were 694 ,g/ml at 0 to 2 h for 250 mg and 1,560 ,ug/ml at 0 to 2 h for 500 mg, and decreased thereafter. The majority of the 24-h excretion of FR10024 was obtained within 4 h after each i.m. injection of 250 or 500 mg. The 24-h urinary recovery rate of cephalothin in five volunteers after i.m. injection was as high as 51.2% for 500 mg; i.e., it was lower than that of FR10024. Biliary excretion. (i) Rats. The 24-h biliary excretion rate of FR10024 after i.m. injection of 20 mg/kg was compared with that of the other cephalosporins (Table 13). The biliary excretion of FR10024 was especially high; 63.3% of the administered dose was excreted in the bile within 24 h after administration. The biliary

ANTIMICROB. AGENTS CHEMOTHER.

NISHIDA ET AL.

60

TABLE 10. Urinary excretion of FR10024 and other cephalosporins in rats after i.m. injectiona Total 6-24 h 3- h 0-3 h Antibiotic pg/ml

mg/ml

9

pg/mi

%

mg

%

59.2 (16.9) 171 (24.7) 83.6 (12.6) 12.2 (3.2) 20.7 (8.2) 51.2

1.42 (0.31) 4.05 (0.50) 2.20 (0.30) 0.30 (0.07) 0.39

0.92 (0.53) 0.44 (0.09) 0.31 (0.05) 0.02 (0.01) 0.04 (0.02) 0.07

1.18 (0.07) 3.59 (0.06) 3.54 (0.12) 0.95 (0.06) 1.52 (0.08) 1.86

28.6 (1.9) 80.2 (1.7) 82.2 (2.2) 22.1 (1.5) 35.1 (2.1) 41.7

(16.6)

(0.40)

8.10 (4.88) 4.04 (0.90) 2.02 (0.35) 0.25 (0.15) 0.28 (0.15) 0.46 (0.23) 190 to 240

%

FR10024

26.3 1,060 (2.0) (214) 75.7 1,580 Cefazolin (1.9) (195) 79.7 1,430 Cephaloridine (2.1) (125) 21.8 395 Cephalothin (1.5) (60.4) 34.7 570 Cephapirin (2.1) (102) 40.5 1,030 Cephacetrile (222) (1.1) a Dose: 20 mg/kg, i.m. Rats: SD strain, parentheses are standard errors.

(0.16) 1.16

male, 6 weeks old,

(1.0) (0.08) (0.03) g, 5 to 10/group. Numbers in

TABLE 11. Urinary excretion of FR10024 and other cephalosporins in monkeys after i.m. injectiona 2-4 h

0-2 h

Total

6-24h

4-6 h

Antibiotic % % mg % % jug/ml p.g/ml % ,ug/ml 76.6 0.43 110 9.2 153 1.38 13.3 839 61.5 1,420 (4.3) (4.4) (0.18) (5.3) (0.70) (48.3) (2.6) (200) (4.9) (341) 80.3 48.6 2.2 120 4.6 28.6 139 45.0 1,240 Cefazolin 1,410 (0.5) (10) (0.7) (17.5) (0.7) (38) (6.8) (441) (8.3) (139) 74.4 115 0.3 218 2.2 6.3 764 58.6 780 15.8 Cephaloridine (0. 1) (1.5) (10) (2.0) (0.6) (3.2) (75) (234) (2.9) (37) 77.2 49.4 0.25 0.02 0.83 9.8 25.4 38.8 405 602 Cephalothin (2.7) (8.2) (0.01) (0.03) (0.27) (9.1) (4.1) (63.3) (4.7) (92) a Dose: 20 mg/kg, i.m. Monkeys: rhesus monkeys, male, 6.4 to 9.9 kg, three/group. Numbers in parentheses are standard errors.

pg/ml

FR10024

TABLE 12. Urinary excretion ofFR10024 and cephalothin in healthy volunteers after i.m. injection Volunteer

Antibiotic

wegt

kg)

FR10024 (250 mg/man)

FR10024 (500

mg/man)

Cephalothin (500 mg/

man)

a

pg/ml

4-6 h

2-4 h

0-2 h

Total

8-24 h

6-8 h

%

pg/ml

%

,ug/ml

%

g/mil

%

p,g/ml

%

mg

%

A (63) F (62) 1 (64) H (56) S (65)

749 255 794 871 802

68.9 53.6 79.4 78.0 77.0

294 202 97.2 80.0 227

20.0 18.6 12.6 7.7 12.2

42.0 42.0 13.3 36.6 34.5

1.5 1.7 1.9 1.0 1.5

2.0 3.2 2.3 5.6 6.5

0.3 0.3 0.3 0.2 0.3

0.4 0.5 0.3 0.6 1.0

0.2 0.2 0.1 0.2 0.3

228 186 236 218 228

90.9 74.4 94.3 87.4 91.3

Mean SEa

694 100

71.4 4.3

180 36

14.2 2.0

33.7 4.7

1.5 0.1

3.9 0.8

0.3 0.02

0.6 0.1

0.2 0.03

219 8

87.7 3.1

A (63) F (62) 1 (64) H (56) S (65)

2,080 762 1,210 1,580 2,180

85.0 57.2 76.3 79.2 77.6

168 290 253 367 805

8.2 11.3 14.4 8.2 15.0

24.7 37.3 17.7 73.6 105

0.76 1.70 0.83 1.10 2.66

3.19 8.70 16.0 10.5 17.0

0.12 0.40 0.94 0.22 0.44

0.61 1.29 1.80 0.99 0.93

0.11 0.29 0.14 0.13 0.15

471 355 462 444 479

94.2 70.9 92.6 88.9 95.9

Mean SE

1,560 238

75.1 4.2

377 100

11.4 1.3

51.7 14.7

1.41 0.32

11.1 2.3

0.42 0.13

1.12 0.18

0.16 0.03

442 20

88.5 4.1

A (63) F (62) 1 (64) H (56)

842 1,220 737 1,970

8.4 9.0 7.3 7.9 9.9

1.1 1.2 0.6 0.7 2.4

3.8 8.7 2.7 6.7 35.7

0.13 0.23 0.11 0.11 0.64

0.12 0.36

0.02 0.04

617

420 565 243 377 551

45.9 55.2 33.9 41.4

S (65)

43.0 36.6 40.6 51.2 35.8

0.30 1.90

0.04 0.24

263 235 243 300 245

52.7 47.1 48.6 58.6 49.0

Mean SE

1,080 219

41.4 2.5

431 53

8.5 0.4

1.2 0.3

11.5 5.5

0.24 0.09

0.54 0.31

0.07 0.04

257 10

51.2 1.8

SE, Standard error.

104

56.1 11.2

61

LABORATORY EVALUATION OF FR10024

VOL. 11, 1977

excretion rate of cefazolin was about 10%. For the other cephalosporins, the biliary excretion rate was 1.6% for cephapirin and less than 1% for cephaloridine, cephalothin, and cephacetrile. (ii) Dogs. The 24-h biliary excretion rate of FR10024 in beagle dogs after i.m. injection of 20 mg/kg was 9.6% (Fig. 6). In this experiment, the biliary excretion of FR10024 was not compared with that of other antibiotics; however, the biliary excretion of other cephalosporins in dogs is known to be comparatively low. The biliary excretion ofFR10024 was approximately three times higher than that reported previously for cefazolin. The biliary levels of FR10024 in dogs were 1,770 ,ug/ml at 1 h and 215 ,ug/ml at 6 h after administration. Antibacterial substances in urine and bile. Using thin-layer chromatography-bioautography, the bioactive substances in the urine and bile of rats, monkeys, and healthy volunteers were investigated after i.m. injection of FR10024 (Fig. 7). Spots of bioactive substances in the urine or bile of each animal and of humans were detected only at the positions corresponding to the standard reference for FR10024. Tissue distribution. Table 14 shows the tissue levels of FR10024 in rats after i.m. injection of 20 mg/kg. FR10024 was distributed into all the tissues 30 min after administration. In the case of the tissue distribution of FR10024, it was found that hepatic levels were higher than those of the other cephalosporins. At 30 min the hepatic levels were 24.9 ug/g for FR10024, 16.3 ,ug/g for cefazolin, and 8.6 ug/g for cephaloridine. Renal levels were 70.3 ,ug/g for cephaloridine, 45.4 p.g/g for cefazolin, and 28.4 ,ug/g for FR10024. Renal levels of the other cephalosporins were all lower than those of FR10024. Other tissue levels, such as of the lungs, heart, and spleen, were the highest for cefazolin, fol-

200r1770 ac/m1

1500 BILIARY

EXCRETION IN

24

HR

9.6 i 1.1 x

-j 3c "I (D

a

w -j

ul w

1050

1000

)/-1

-i

m

4 .-i

40' 600 )Wc1

5001-

457

-WJ1

215 ,aWml

3

1

4

5

6

TIME (HR)

FIG. 6. Biliary levels ofFR10024 in five dogs after i.m.

injection.

lowed by cephaloridine, FR10024, cephacetrile, cephapirin, and cephalothin.

DISCUSSION Although Citrobacter, E. cloacae, andE. aerogenes are generally regarded as bacteria that are resistant to cephalosporins, a significant number are susceptible to FR10024 at an inoculum of 106 cells/ml. Clinical studies will be necessary to confirm whether the antibacterial activity of FR10024 against these strains is of

TABLE 13. Biliary excretion of FR10024 and other cephalosporins in rats after i.m. injectiona Antibiotic

0-3 h

3-6 h

Ag/ml 9 &g/9ml 939 61.8 31.0 1.42 (104) (6.7) (5.2) (0.24) Cefazolin 141 9.4 12.3 0.50 (17.1) (1.3) (1.3) (0.05) Cephaloridine 6.0 0.37 1.23 0.05 (0.72) (0.05) (0.14) (0.01) Cephalothin 12.0 0.90 0.05 0.00 (1.8) (0.14) (0.02) (0.00) 22.9 Cephapirin 1.59 0.26 0.01 (1.3) (0.13) (0.07) (0.00)

Laboratory evaluation of FR10024 a new cephalosporin derivative.

Vol. 11, No. 1 Printed in U.S.A. ANTIMICROBIAL AGENTS AND CHEICOTHERAPY, Jan. 1977, p. 51-63 Copyright © 1977 American Society for Microbiology Labo...
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