Antipneumococcal activity of MEN 10700, a new penem, compared with other compounds, by MIC and time–kill kinetics

Glenn A. Pankucha, Dianne B. Hoellmana, Michael R. Jacobsb and Peter C. Appelbauma,*

a Department of Pathology (Clinical Microbiology), Hershey Medical Center, 500 University Drive, Hershey, PA 17033 b Case Western Reserve University, Cleveland, OH 44106, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antipneumococcal activity of MEN 10700 was compared with those of nine other compounds by MIC and time–kill kinetics. MIC90s (mg/L) of 202 penicillin-susceptible, -intermediate and -resistant pneumococci were: 0.06, 1.0 and 2.0 (MEN 10700); 0.06, 0.5–1.0 and 2.0 (amoxycillin ± clavulanate); 0.06, 0.5 and 4.0 (cefotaxime); 0.125, 0.5 and 2.0 (cefepime); 0.016, 0.125 and 0.25 (imipenem); 0.03, 0.5 and 1.0 (meropenem); 2.0 (ciprofloxacin); 0.125, > 64.0 and > 64.0 (clarithromycin); and 0.5 (vancomycin). Time–kill kinetics showed that MEN 10700, at 4 x MIC, was bactericidal for all 12 isolates tested at 4 x MIC. Kinetics of other ß-lactams were similar to those of MEN 10700, relative to MICs. Ciprofloxacin, at 4 x MIC, was uniformly bactericidal after 24 h. Clarithromycin exhibited slow kill kinetics, after 24 h. Vancomycin was bactericidal against 11/12 isolates at 2 x MIC after 24 h.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The world-wide incidence of infections caused by pneumococci resistant to penicillin G and other antimicrobials has increased at an alarming rate during the past two decades, and in particular during the past 5 years.1 There is a need for a broad-spectrumß-lactam agent for oral and parenteral treatment of resistant pneumococcal infections in children and adults.

MEN 10700 is a new alkylamino penem with a sarcosinamido group at position 2.2,3 Previous reports have documented the broad-spectrum activity of this compound against a wide variety of aerobic and anaerobic Gram-positive and -negative organisms, including pneumococci.2,3 This study expands the latter findings by testing (i) the activity of MEN 10700, compared with amoxycillin, co-amoxiclav, cefotaxime, cefepime, imipenem, meropenem, ciprofloxacin, clarithromycin and vancomycin against 202 penicillin susceptible and resistant pneumococci by agar dilution; (ii) the activity of the same compounds against 12 penicillin susceptible and resistant pneumococci by time–kill kinetics.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates and antimicrobials

Two hundred and two pneumococcal isolates were studied by agar dilution, and comprised 57 penicillin-susceptible (MIC <= 0.06 mg/L), 84 -intermediate (MIC 0.125–1.0 mg/L) and 61 -resistant (MICs >= 2.0 mg/L) isolates. For time–kill kinetics, four each of penicillin-susceptible, -intermediate and -resistant isolates were studied. MEN 10700 was obtained from Menarini Ricerche, S.p.A. (Florence, Italy). Other compounds were obtained from their respective manufacturers.

MIC method

Agar dilution for the 202 isolates was performed in air as described previously by our group1 using Mueller–Hinton agar (BBL) with 5% added sheep blood and inocula of 1 x 104 cfu/spot. NCCLS does not recommend agar dilution for routine pneumococcal susceptibility testing. Broth MICs for the 12 isolates tested by time–kill were determined, according to NCCLS recommendations, in Mueller–Hinton broth with 5% added lysed horse blood and inocula of 1 x 105 cfu/mL.4 Clavulanate was added to amoxycillin at a fixed ratio of 1:2. Standard quality control isolates4 were included in each run of agar and broth dilution MICs. Because isolates tested were stock isolates which had been subcultured many times, CO2 was not needed for adequate growth.

Time–kill testing

Tubes containing 5 mL cation-adjusted Mueller–Hinton broth + 5% lysed horse blood with doubling antibiotic concentrations were inoculated with 5 x 105 to 5 x 106 cfu/mL and incubated at 35°C in a shaking water bath. The original inoculum was determined by using the untreated growth control. Viability counts of antibiotic-containing suspensions were performed at 0, 3, 6, 12 and 24 h, by plating dilutions onto 5% sheep blood agar plates. The lower limit of sensitivity of colony counts was 300 cfu/mL.4

Time–kills were analysed by determining the number of isolates yielding a difference in log10 cfu/mL of –1, –2 and –3 at 0, 3, 6, 12 and 24 h, compared with counts at time 0 h. Antimicrobials were considered bactericidal at the lowest concentration that reduced the original inoculum by >3 log10 cfu/mL (99.9%) at each of the time periods, and bacteriostatic if the inoculum was reduced by 0–3 log10 cfu/mL. With sensitivity threshold and inocula used in these studies, no problems were encountered in delineating 99.9% killing, when present. Bacterial carryover was addressed by dilution as described previously.4


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Agar dilution MICs for the 202 isolates are presented in Table I. As can be seen, MICs of MEN 10700 against penicillin-susceptible, -intermediate and -resistant isolates were similar to those of amoxycillin, co-amoxiclav, cefotaxime, cefepime and meropenem; imipenem yielded the lowest MICs against all three groups. Ciprofloxacin and vancomycin MICs were unaffected by penicillin MICs, and clarithromycin resistance was mainly found in penicillin-intermediate and -resistant isolates.


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Table I. Agar dilution MICs (mg/L) of a variety of antimicrobial agents against 202 isolates of S. pneumoniae
 
Broth dilution MICs of the 12 isolates tested by time–kill were all within one dilution of those obtained by agar dilution. Results of time–kill assays are presented in Table II. As can be seen, MEN 10700 was bactericidal (99.9% killing) after 24 h against all 12 isolates at 4 x MIC, with 99% killing of all isolates at 2 x MIC after 12 h and 24 h. After 6 h, MEN 10700 showed 90% killing of all 12 isolates at 2 x MIC, with 90% killing of 11/12 isolates at 8 x MIC after 3 h. Kill kinetics of other ß-lactams tested, relative to their MICs, were all similar to that of MEN 10700. Ciprofloxacin, at 4 x MIC, was bactericidal against all isolates after 24 h. Against the eight clarithromycin susceptible isolates, clarithromycin exhibited slow kill kinetics, with 99.9% killing of 6/8 isolates after 24 h at 4 x MIC. Vancomycin was bactericidal against 11/12 isolates at 2 x MIC after 24 h.


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Table II. Results of time–kill testing of 12 isolates
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The penem nucleus may be regarded as a hybrid of penicillin (as in penicillins) and cephem (as in cephalosporins). Penems were first described about 20 years ago, but the first commercially oriented compounds in this class, SCH 29482 and SCH 34343, did not progress past preliminary stages of evaluation.5,6 Penems and carbapenems that have been described recently and are under varying stages of development in different countries are faropenem (SUN 5555), ritipenem (FCE 22101), sulopenem (CP 65207), CS 834 and DU-6681. 3 ,7 ,8 ,9 ,10

MEN 10700 is another recently described novel broad-spectrum penem.2,3 In a recent study,3 MEN 10700 was found to be most active against staphylococci and streptococci, slightly less active against Escherichia coli, Klebsiella pneumoniae, Enterobacterspp., Citrobacter spp., Moraxella catarrhalis and peptostreptococci, moderately active against Enterococcus faecalis, Proteus spp., Serratia marcescens, Acinetobacter spp., Clostridium and Bacteroides spp., and inactive against Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Enterococcus faecium.In another study, Fontana and co-workers2 found that the antibacterial spectrum of MEN 10700 was slightly narrower than that of imipenem, but compared favourably with those of ritipenem and cefotaxime. In preliminary studies included in the latter two papers,2,3 MIC50/MIC90 values of MEN 10700 were 0.015–0.03/0.03–0.06 and 0.5/1.0 mg/L against penicillin-susceptible and -resistant isolates, respectively. However, the penicillin MICs for the latter group were not given. Bactericidal activity was demonstrated by the replica plating method.

In our study, MEN 10700 was found to have MICs comparable with those of amoxycillin, co-amoxiclav, cefotaxime, cefepime and meropenem.1 Imipenem had the lowest MICs of all ß-lactams tested, but the epileptogenic potential of this compound1 precludes its use in serious systemic pneumococcal infections. Meropenem seems to lack this side-effect and may be used for treatment of these infections. Choice between meropenem and a penem such as MEN 10700 should be made upon the basis of MICs in combination with pharmacokinetic and pharmacodynamic studies (not yet available for MEN 10700).

Kill kinetics of MEN 10700 were similar to those of all other ß-lactams studied, relative to MICs. Because MICs of ciprofloxacin cluster around the breakpoint, it is not recommended for treatment of serious pneumococcal infections.1 Slow kill kinetics of macrolides against pneumococci, as well as higher macrolide MICs in pneumococci with raised penicillin MICs, have been described.4

If results of toxicological and pharmacokinetic studies show promise, this study indicates that MEN 10700 shows potential for treatment of infections caused by penicillin-susceptible and -resistant pneumococci.


    Acknowledgments
 
This study was supported by a grant from Menarini Ricerche S.p.A., Florence, Italy.


    Notes
 
* Corresponding author. Tel: +1-717-531-5113; Fax: +1-717-531-7953; E-mail: pappelbaum{at}psghs.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Jacobs, M. R. & Appelbaum, P. C. (1995). Antibiotic-resistant pneumococci. Reviews of Medical Microbiology 6, 77–93.

2 . Altamura, M., Perrotta, E., Sbraci, P., Pestellini, V., Arcamone, F., Cascio, G. et al. (1995). 2-Substituted penems with amino acid-related side chains: synthesis and antibacterial activity of a new series of ß-lactam antibiotics. Journal of Medical Chemistry 38, 4244–56.

3 . Hamilton-Miller, J. M. T. & Shah, S. (1997). In-vitro microbiological assessment of a new penem, MEN 10700. Journal of Antimicrobial Chemotherapy 39, 575–84.[Abstract]

4 . Pankuch, G. A., Lichtenberger, C., Jacobs, M. R. & Appelbaum, P. C. (1996). Antipneumococcal activities of RP 59500 (quinupristin/dalfopristin), penicillin G, erythromycin and sparfloxacin determined by MIC and rapid time–kill methodologies. Antimicrobial Agents and Chemotherapy 40, 1653–6.[Abstract]

5 . Ganguly, A. K., Afonso, A., Girijavallabhan, V. M. & McCombie, S. (1985). Synthesis and preliminary in-vitro profile of Sch 34343—a new penem antibacterial agent. Journal of Antimicrobial Chemotherapy 15, Suppl. C, 1–4.[ISI][Medline]

6 . Phillips, I., Wise, R. & Neu, H. C. (1982). An oral penem antibiotic: Sch 29482. Journal of Antimicrobial Chemotherapy 9, Suppl. C, 1–247.[ISI][Medline]

7 . Inoue, M. & Mitsuhashi, S. (1994). In vitro antibacterial activity and ß-lactamase stability of SY5555, a new oral penem antibiotic. Antimicrobial Agents and Chemotherapy 38, 1974–9.[Abstract]

8 . Tanaka, M., Hohmura, M., Nishi, T., Sato, K. & Hayakawa, I. (1997). Antimicrobial activity of DU-6681a, a parent compound of novel oral carbapenem DZ-2640. Antimicrobial Agents and Chemotherapy41 , 1260–8.[Abstract]

9 . Utsui, Y., Takenouchi, T., Masuda, N., Domon, H., Kakuta, M., Ishii, C. et al. (1996). CS-834, a new oral carbapenem: II. In vitro evaluation of R-95867, the active metabolite of CS-834. In Program and Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1996. Abstract F106, p. 118. American Society for Microbiology, Washington, DC.

10 . Wise, R., Andrews, J. M. & Danks, G. (1983). Comparison of in vitro activity of FCE 22101, a new penem, with those of other ß-lactam antibiotics. Antimicrobial Agents and Chemotherapy 24, 909–14.[ISI][Medline]

Received 12 November 1998; returned 10 February 1999; revised 24 February 1999; accepted 6 April 1999





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