ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1976, p. 543-548 Copyright C 1976 American Society for Microbiology

Vol. 10, No. 3

Printed in U.S.A.

5"-Amino-5"-Deoxybutirosin, a Semisynthetic Analogue of Butirosin A: Antibacterial Activity In Vitro and in Mice C. L. HEIFETZ,* C. A. FREY,

I.

A. PEARSON,

AND

J. C. SESNIE

Biological Research and Development Department, Research and Medical Affairs Division, Parke, Davis & Company, Detroit, Michigan 48232 Received for publication 21 May 1976

Aminodeoxybutirosin (AD-BTN), the 5"-amino-5"-deoxy derivative of butirosin (BTN), was synthesized to improve on the antibacterial activity of BTN by preventing bacterial enzymatic phosphorylation at the 5' position. AD-BTN possesses the spectrum characteristic of BTN and gentamicin (GTM) and was active at low levels in vitro against a wide variety of gram-negative species including Pseudomonas aeruginosa, indole-positive Proteus and Serratia marcescens; its action was bactericidal against both light and heavy inocula, and it was not antagonized by human serum. AD-BTN was as active as GTM against GTM-sensitive P. aeruginosa in vitro and in mice, and was markedly improved over BTN. AD-BTN retained the good activity of the parent compound against other gram-negative pathogens. Whereas GTM minimal inhibitory concentrations were elevated 35-fold against GTM-resistant P. aeruginosa in vitro, the minimal inhibitory concentration of AD-BTN was only doubled. At 6.3 ,ug/ml, AD-BTN inhibited 68% of 82 isolates insusceptible to that concentration of GTM. In murine toxicity tests AD-BTN was about one-third to one-half as toxic as GTM. tion method with a synthetic TB medium was used (3). All incubations were aerobic at 37°C for 16 to 18 h (except M. tuberculosis which was incubated for 2 weeks). Bacteria manifesting MICs of BTN >25 ,ug/ ml or MICs of GTM >6.3 ug/ml were classified as resistant (5). To determine minimal bactericidal concentrations (MBCs), a microtitration procedure was used (4). After microtitration MICs were determined the presence of residual viable bacteria in the antiMATERIALS AND METHODS biotic-containing wells was assessed by subculturThe antibiotics were handled, evaluated, and re- ing 0.05-ml volumes onto MHA plates, using microported in terms of base content. We used butirosin titer loops for sampling. Tests in vivo were done in 18- to 20-g female CD-1 sulfate (BTN) containing 710 ltg of base per mg and gentamicin sulfate (GTM) containing 561 ,g of base mice (Charles River). They received mouse laboraper mg. The latter was obtained through the gener- tory chow (Purina) and water ad libitum. Subcutaosity of Marvin J. Weinstein of the Schering Corp. neous (SC) injections were in 0.5-ml volumes of The 5"-amino-5"-deoxybutirosin sulfate (AD-BTN), physiological saline and intravenous doses were 0.25 containing 688 ,ug of base per mg, was derived from ml in distilled water. To determine acute drug toxicities in mice, single the A component of butirosin, because the B compo1.5-fold graded doses of compound were adminisnent derivative was found to be less active (1). Minimal inhibitory concentrations (MICs) were tered SC and intravenously to groups of 10 mice. The determined by previously published procedures (5). subacute toxicity of AD-BTN in mice was ascerAgar dilution in standardized Mueller-Hinton agar tained by injecting 1.5-fold graded doses of SC twice (MHA) was performed according to the recom- daily for 5 days. Because no deaths occurred after mendations of the international collaborative study the second day, the tests were not extended for an (2). Approximately 104 colony-forming units of bac- additional 5 days as had been done previously with teria were applied to the surface of a plate with a BTN and GTM (5). The median lethal doses (LD50's) Steers replicator (9). Trypticase soy broth (TSB) or were estimated by log-probit analysis (8), using TSB with 10% bovine serum were used in the micro- pooled percent mortality data from at least two titration procedure (7); inocula contained approxi- tests. Protective activities of AD-BTN, BTN, and GTM mately 2,000 colony-forming units in 0.05-ml volumes. For Mycobacterium tuberculosis, a tube dilu- were compared in standard acute mouse protection 543

Culbertson, Watson, and Haskell synthesized 5"-amino-5"-deoxybutirosin as a means of preventing the enzymatic phosphorylation ofthe 5" position of butirosin, and its inactivation, by resistant bacteria (1). Reported herein are studies regarding the in vitro and mouse-protective antibacterial activities of this semisynthetic butirosin analogue.

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tests (5). Single SC doses in twofold rising incremental series were administered concurrently with bacterial challenge. Challenges were accomplished by the intraperitoneal injection of an estimated 100 LD50 suspended in 0.4 ml of 5% hog gastric mucin or 0.5 ml of TSB (Klebsiella only). Generally 590% of the untreated controls died within 48 to 72 h. Final survival percentages, obtained after 7 days of observation among groups of 8 to 15 mice, were pooled and used to estimate median protective doses (PD50) by the log-probit method (8).

RESULTS

The antibacterial spectrum of AD-BTN in vitro is delineated in Table 1. The antibiotic was active at low levels against a wide variety of clinically important gram-negative species including Pseudomonas aeruginosa and indolepositive Proteus species (P. morganii, P. rettgeri, P. vulgaris, and Providencia). Also susceptible were Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Protus mirabilis, various Salmonellae and Shigellae, and Serratia marcescens.

Activity against other species were variable. Although inhibitory against M. tuberculosis, Staphylococcus epidermidis, and strains of S. aureus with limited antibiotic resistance, ADBTN was weak or inactive against pneumococci, streptococci, and multi-antibiotic resistant S. aureus. The bacteriostatic and bactericidal activities of AD-BTN, BTN, and GTM versus light and heavy bacterial inocula of single strains of five bacterial species are compared in Table 2. With one exception, the MICs and MBCs of AD-BTN were equal. This finding indicates a possible superiority in bactericidal potency over the parent compound for which the MBCs were twice the MICs against heavy inocula. A 1,000-fold increase in inocula generally produced only a doubling of MICs and MBCs of AD-BTN. None of the compounds was appreciably antagonized by serum. In the presence of 25% human serum in TSB, microtitration MICs of AD-BTN, BTN, and GTM were the same or 1 dilution lower than the MICs in serum-free

TABLE 1. Antibacterial spectrum of aminodeoxybutirosin Species

No. of strains tested

MIC (y.g/ml)

Test methoda

Range

Geometric mean

Enterobacter cloacae 5 MHA 1.6 1.6 Escherichia coli 9 MHA 1.6-3.1 2.0 Klebsiella pneumoniae 10 MHA 0.4-1.6 0.9 Proteus mirabilis 2 MHA 3.1-12.5 6.3 P. morganii 1 MHA 1.6 1.6 P. rettgeri 2 MHA 1.6-3.1 2.2 P. vulgaris 2 MHA 6.3 6.3 Providencia sp. 6 MHA 1.6-25 14.7 Pseudomonas aeruginosa 47 MHA 0.8-12.5 3.8 Salmonella enteritidis 2 MHA 1.6 1.6 S. minnesota 1 MHA 1.6 1.6 S. paratyphi 1 MHA 0.4 0.4 S. typhimurium 1 MHA 3.1 3.1 S. typhosa 4 TSB 1.6-6.3 5.2 Serratia marcescens 6 MHA 1.6-12.5 3.5 Shigella dysenteriae 2 MHA 3.1-6.3 4.4 S. fZexneri 4 MHA 3.1-6.3 5.3 S. sonnei 3 MHA 1.6-3.1 2.5 Staphylococcus aureus Antibiotic sensitive 1 TSB 0.8 0.8 Limited resistanceb 2 TSB 12.5-25 17.7 Macrolide resistant 1 TSB 0.8 0.8 Methicillin resistant 1 TSB 1.6 1.6 Multiresistant 1 TSB 50 50 S. epidermidis 2 TSB 0.8-1.6 1.3 Streptococcus faecalis 1 TSB + S >100 >100 S. pneumoniae 1 TSB + S 100 100 S. pyogenes 1 TSB + S 50 50 1 Mycobacterium tuberculosis TB 5 5 a TSB and TSB + S, Microtitration dilution in TSB and TSB with 10% bovine serum; TB, tube dilution in synthetic TB medium. b Phage type 80/81 strains resistant to penicillin and streptomycin, and susceptible to neomycin, kanamycin, and paromomycin.

AD-BTN ANTIBACTERIAL ACTIVITY

VOL. 10, 1976

broth, when tested against P. aeruginosa (strain no. 28) and S. aureus (strain UC-76). Antibacterial spectrums of AD-BTN, BTN, and GTM against several GTM-sensitive bacteria (GTM MIC 25 ,ug/ml) organisms as possible in this comparison.

With the exception of S. marcescens, ADBTN and BTN possessed approximately equivalent mouse-protective activities against BTNsusceptible Enterobacteriaceae. As expected, the PDH0's of BTN were much higher against BTN-resistant isolates; AD-BTN was several times more potent than BTN against these bacteria. Against the two strains resistant to BTN but sensitive to GTM in vitro, AD-BTN was at least as effective as the latter in mice. However, against strains resistant to both BTN and GTM in vitro, the latter was more effective

TABiE 5. Comparative mouse toxicities of aminodeoxybutirosin, butirosin, and gentamicin LD50 ± 95% confidence limits (mg of base/kg)

Toxicity test AminodeoxyButirosin Gentamicin butirosin Single intravenous 126 ± 7 50 ± 6 460 ± 14 dose Single SC dose 1420 ± 158 6000 ± 470 410 + 12 Subacute SC dose 770 ± 118 4260 ± 422 340 ± 89

than AD-BTN in five of eight instances. The geometric mean PD50's of AD-BTN and GTM versus all BTN-resistant Enterobacteriaceae were 50 and 26 mg of base per kg, respectively. Among isolates of P. aeruginosa, the relationships between in vitro susceptibilities and responses in mouse protection tests were unpredictable. This finding may have derived from the fact that many of the bacteria lie on the borderline between in vitro susceptibility and resistance to BTN and/or GTM. However, some generalizations may be drawn. Against apparently BTN-susceptible strains, the aminodeoxy derivative was three to four times more active than the parent compound and was equally as effective as GTM. The geometric mean PD5o's of AD-BTN, BTN, and GTM were 23, 76, and 21 mg of base per kg, respectively, against these 14 P. aeruginosa strains. Against the 8 BTNresistant varieties, the same general relationship existed between the three antibiotics, at an approximate sevenfold diminished level of activity. Thus, the geometric mean PD50's of

VOL. 10, 1976

AD-BTN ANTIBACTERIAL ACTIVITY

AD-BTN, BTN, and GTM, respectively, were 158, 519, and 157 mg of base per kg. DISCUSSION By preparing the 5"-amino-5"-deoxy derivative, Culbertson, Watson, and Haskell (1) attempted to improve upon the antibacterial ac-

547

tivity of BTN, a compound noted for its low toxicity and for its activity against approximately half of the GTM-resistant isolates against which it had been tested (5, 6). They attempted to accomplish such improvement by preventing enzymatic phosphorylation at the 5" position of BTN.

TABLz 6. Comparative in vitro and mouse-protective activities of aminodeoxybutirosin, butirosin, and gentamicin versus Enterobacteriaceae AminodeoxyButirosin Gentamicin butirosin Gnaii Test organism MIC MIC MIC PD50 PD,50 PDSO (,ug/ml) (mg/kg) (,ugmIl) (mg/kg) (,ug/ml) (mg/kg) Enterobactercloacae IMM-1l 1.6 2.7 1.6 2.7 0.8 1.5 Klebsiella pneumoniae MGH-2 1.6 4.1 1.6 5.9 0.8 1.9 Proteus vulgaris UC-232 6.3 4.3 3.1 6.8 1.6 1.7 Serratia marcescens IMM-5 3.1 25 6.3 100 0.4 5.4 Providencia sp. D 288 (BTN-R)a 1.6 22 50 174 3.1 46 Enterobacter sp. B 2716 (BTN/GTM-R) 50 85 100 220 12.5 0.6 Providencia sp. 2328 (BTN-R) 6.3 19 >100 213 6.3 17 Providencia sp. C482 (BTN/GTM-R) 25 27 >100 >710 25 39 K. pneumoniae B 1447 (BTN/GTM-R) 50 80 >100 263 25 35 K. pneumoniae D 1013 (BTN/GTM-R) 100 75 >100 355 22 25 P. morganii C 558 (BTN/GTM-R) 25 100 >100 >710 25 56 P. rettgeri B1796 (BTN/GTM-R) 12.5 49 >100 375 12.5 59 P. rettgeri B 2038 (BTN/GTM-R) 12.5 100 >100 994 12.5 67 P. rettgeri C 941 (BTN/GTM-R) 6.3 35 >100 852 12.5 40 a BTN-R, Butirosin resistant; GTM-R, gentamicin resistant; BTN/GTM-R, resistant to both. TABLE 7. Comparative in vitro and mouse-protective activities of aminodeoxybutirosin, butirosin, and gentamicin versus P. aeruginosa Aminodeoxybutirosin

P. aeruginosa isolate

MIC

PD5o

Butirosin MIC PD5o

Gentamicin MIC

(,ug/ml) (mg/kg) (,ug/ml) (mg/kg) (,ug/ml) Bernardi 0.8 18 3.1 18 0.8 SD-1C 0.8 3.1 6 0.8 9 SD-5C 0.8 18 3.1 57 0.8 Siebart 1.6 62 3.1 87 0.8 SD-4B 1.6 36 6.3 82 0.8 SD-4C 0.8 31 1.6 6.3 53 SD-7A 0.8 37 1.6 6.3 74 SD-7B 0.8 18 6.3 85 1.6 72 (GTM-R)a 3.1 12.5 25 11 43 Assemany 3.1 40 3.1 25 89 SD-3B 1.6 31 25 >710 0.8 UI-18 3.1 25 3.1 27 65 VAD-12-7-7 3.1 30 25 1.6 82 G 75 (GTM-R) 12.5 110 25 362 200 1174 (BTN-R) 12.5 210 50 6.3 568 71 (BTN/GTM-R) 12.5 100 50 376 100 G 30 (BTN/GTM-R) 6.3 135 200 259 50 G76 (BTN/GTM-R) 12.5 92 200 50 259 PIU PMG 5 (BTN-R) 100 >344 100 >710 1.6 Moeller 13485 (BTN/GTM-R) 6.3 34 100 128 25 Bold 6-22 (BTN/GTM-R) 50 88 >710 >100 50 Jenkins (BTN/GTM-R) 50 >355 >100 >710 50 a BTN-R, Butirosin resistant; GTM-R, gentamicin resistant; BTN/GTM-R, resistant to both.

PD50 (mg/kg) 8 2.7 7 17 22 13 18 13 84 32 15 32 23 >280 163 >280 >280 >280 12 12 170 >266

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AD-BTN demonstrated some superiority over BTN with respect to relative bactericidal to baeteriostatic potency against heavy inocula. Both in vitro and in mice, this analogue was clearly more potent than BTN against P. aeruginosa and S. marcescens, whereas it retained the good activity of BTN against other gram-negative bacterial pathogens. AD-BTN was almost as active as GTM against GTMsensitive isolates of P. aeruginosa and was active against a majority of GTM-resistant strains of P. aeruginosa. As often appears to be the case with aminoglycoside antibiotics, the price of improved antibacterial potency was increased toxicity. Thus, AD-BTN was found to be about four times as toxic as BTN in acute intravenous and SC tests in mice, and about 5.5 times as toxic in subacute SC tests. How these observations relate to more pertinent chronic renal and ototoxicity in other species or possible tolerated doses in man has not been determined. LITERATURE CITED 1. Culbertson, T. P., D. R. Watson, and T. H. Haskell. 1973. 5"-Amnho-5!'-deoxybutirosin, a new semi-syn-

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thetic aminoglycoside antibiotic. J. Antibiot. 26:790793. Ericsson, H., and J. C Sherria. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. 217 (Suppl.):64-73. Fisher, M. W., M. C. Manning, L. A. Gagliard, M. R. Gaetz, and A. L. Erlandson. 1960. Paromomycin: experimental antibacterial activity. Antibiot. Annu. 1959-1960, p. 293-303. Harwick, H. J., P. Weiss, and F. R. Fekety, Jr. 1968. Application of microtitration techniques to bacteriostatic and bactericidal antibiotic susceptibility testing. J. Lab. Clin. Med. 72:511-516. Heifetz, C. L., J. A. Chodubski, I. A. Pearson, C. A. Silverman, and M. W. Fisher. 1974. Butirosin compared with gentamicin in vitro and in vivo. Antimicrob. Agents Chemother. 6:124-135. Heifetz, C. L., M. W. Fisher, J. A. Chodubski, and M. 0. DeCarlo. 1972. Butirosin, a new aminoglycoside antibiotic complex: antibacterial activity in vitro and in mice. Antimicrob. Agents Chemother. 2:89-94. Marymont, J. H., Jr., and R. M. Wentz. 1966. Serial dilution antibiotic sensitivity testing with the microtitrator system. Am. J. Clin. Pathol. 45:548-551. Miller, L. C., and M. L. Tainter. 1944. Estimation of the ED. and its error by means of logarithmic-probit graph paper. Proc. Soc. Exp. Biol. Med. 57:261-274. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility of antibiotics. Antibiot. Chemother. 9:307-311.

"5-Amino-5"-deoxybutirosin, a semisynthetic analogue of butirosin A: antibacterial activity in vitro and in mice.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1976, p. 543-548 Copyright C 1976 American Society for Microbiology Vol. 10, No. 3 Printed in U.S.A. 5...
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