Therapeutic efficacy of intraperitoneal polymyxin B and polymyxin-like peptides alone or combined with levofloxacin in rat models of septic shock

Andrea Giacomettia, Oscar Cirionia,*, Roberto Ghisellib, Federico Mocchegianib, Alessandra Mataloni Paggia, Fiorenza Orlandoc, Wojciech Kamyszd, Franciszek Kasprzykowskid, Zbigniew Mackiewiczd, Giorgio Scalisea and Vittorio Sabab

a Institute of Infectious Diseases and Public Health, University of Ancona, Ancona; b Department of General Surgery 1, INRCA IRRCS, University of Ancona, Ancona; c Biotechnology Centre, Research Department, INRCA IRRCS, Ancona, Italy; d Faculty of Chemistry, University of Gdansk, Gdansk, Poland


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The efficacy of two polymyxin-like peptides, KFFKFFKFF and IKFLKFLKFL, alone and combined with levofloxacin, was investigated in a rat model of septic shock. Rats were given an ip injection of 2 x 1010 cfu of Escherichia coli and randomized to receive ip isotonic sodium chloride solution, 7 mg/kg levofloxacin, 1 mg/kg polymyxin B and 1 mg/kg of each polymyxin-like peptides alone or combined with 7 mg/kg levofloxacin. Polymyxins achieved a significant reduction in plasma endotoxin and tumour necrosis factor {alpha} (TNF-{alpha}) concentration. Levofloxacin significantly reduced the bacterial growth and TNF-{alpha} concentration. The combinations of polymyxin-like peptides and levofloxacin demonstrated the highest efficacy.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Septic shock remains a serious condition with a high mortality rate.1 The endotoxin or lipopolysaccharide (LPS) associated with cell membranes of Gram-negative bacteria is its major pathogenic factor. It is composed of core polysaccharide and a lipophilic fatty acid. Core polysaccharide consists of a short-chain of sugars. Two unusual sugars are normally present, heptose and 2-keto-3-deoxyoctonoic acid. Lipid A is the lipid component of LPS. It contains the hydrophobic membrane-anchoring region of LPS. It consists of a phosphorylated N-acetylglucosamine (NAG) dimer with six or seven saturated fatty acids (FA) attached. Some FA are attached directly to the NAG dimer and others are esterified to 3-hydroxy fatty acids. The structure of lipid A is highly conserved among Gram-negative bacteria. Lipid A induces expression of cytokine genes through stimulation of receptors on the surface of target cells.2 In its presence these cells respond by producing numerous inflammatory mediators, such as tumour necrosis factor (TNF), interleukin (IL)-1, platelet-activating factor, arachidonic acid metabolites, erythropoietin, endothelin, IL-6 and IL-8. Several studies have shown an association between TNF levels, severity of shock and fatality.1–3

Polymyxins are peptides produced by Paenibacillus polymyxa. Among polymyxins, only polymyxin B and E (colistin) are used therapeutically. They cross the Gram-negative outer membrane and act on the cytoplasmic membrane, leading to permeability changes and cell death. They also bind LPS and provide protection against LPS-associated mortality.4

KFFKFFKFF and IKFLKFLKFL are novel synthetic cationic peptides that have antibacterial properties. They sensitize Gram-negative bacteria to hydrophobic and amphipathic antibiotics such as rifampicin, erythromycin, novobiocin and fusidic acid.5

Fluoroquinolones have been shown to have an immunomodulatory effect on cytokine production by LPS-treated human monocytes, and have a protective effect in LPS-treated mice.6

The aim of the present study was to investigate the efficacy of ip polymyxin B and polymyxin-like peptides, alone or in combination with levofloxacin, in a rat model of septic shock.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Escherichia coli ATCC 25922 was used as a quality control strain.

Synthetic peptides

Peptides were synthesized using an automatic plus pep synthesizer (Milligen 9050; Millipore Corporation, Burlington, VT, USA) and were purified by reversed-phase high-pressure liquid chromatography (HPLC) on a Knauer two-pump Well-Chrom K1001 system. The products were analysed by HPLC, chemical analysis and fast atom bombardment mass spectrometry.

Drugs

Polymyxin B (Sigma–Aldrich, Milan, Italy), KFFKFFKFF and IKFLKFLKFL (Faculty of Chemistry, University of Gdansk, Poland) were dissolved in distilled water and diluted in physiological saline. Levofloxacin was obtained from Aventis Pharma S.p.A., Milan, Italy.

Animals

Adult male Wistar rats (weight range 250–330g) were used. All animals had access to food and water ad libitum throughout the study. The study was approved by the animal research ethics committee of the INRCA IRRCS, University of Ancona, Ancona, Italy.

Preparation of the inoculum

E. coli ATCC 25922 in the logarithmic phase of growth was centrifuged at 1000g for 15 min. The supernatant was discarded and the bacteria were resuspended in sterile saline to achieve a concentration of 2 x 1010 cfu/mL.

Implantation of inoculum

All animals were anaesthetized by an im injection of ketamine (30 mg/kg) and received an ip inoculum of 1 mL saline containing 2 x 1010 cfu of E. coli ATCC 25922, with the exception of the uninfected control group (C0).

Antibiotic therapy

Four groups of animals were given levofloxacin 7 mg/kg ip, polymyxin B 1 mg/kg, KFFKFFKFF 1 mg/kg and IKFLKFLKFL 1 mg/kg, respectively, immediately after bacterial challenge. In addition, three groups received 1 mg/kg polymyxin B, KFFKFFKFF and IKFLKFLKFL in combination with 7 mg/kg levofloxacin. The study included an untreated control group (C1) and the uninfected group C0.

Plasma endotoxin and TNF-{alpha} levels

Blood samples were collected from the jugular vein 0, 60, 120 and 240 min post-injection. Endotoxin concentrations were measured by a Limulus amoebocyte lysate test (E-TOXATE; Sigma–Aldrich). Endotoxin standards (0, 0.015, 0.03, 0.06, 0.125, 0.25 and 0.5 EU/mL) were tested in each run and the concentration of endotoxin in the test samples was calculated by comparison with the standard curve.

TNF-{alpha} levels were measured with a solid phase sandwich enzyme-linked immunosorbent assay (Nuclear Laser Medicine, S.r.l., Settala, Italy). Samples were compared with the standard curve. All samples were run in duplicate. The lower limit of sensitivity for TNF-{alpha} by this assay was 4 pg/mL.

Evaluation of treatment

Surviving animals were killed with chloroform 48 h post-injection, and blood samples for culture were obtained by aseptic percutaneous transthoracic cardiac puncture. To perform quantitative bacterial evaluation of the intraabdominal fluid, 10 mL of sterile saline was injected ip, samples of the peritoneal lavage fluid were serially diluted and a 0.1 mL volume of each dilution was spread on blood agar plates. Plates were incubated in air at 35°C for 48 h. The limit of detection was <1 log10 cfu/mL.

Statistical analysis

Survival data were compared using log rank test; qualitative results from blood and intra-abdominal fluid cultures were analysed using {chi}2, Yates's correction and Fisher's exact test, depending on the sample size. Quantitative bacterial evaluation of the intra-abdominal fluid cultures were presented as mean ± S.D. of the mean; statistical comparisons between groups were made using ANOVA. Post hoc comparisons were performed by Bonferroni's test. Plasma endotoxin and TNF-{alpha} levels were analysed using the Kruskal–Wallis test; multiple comparisons between groups were performed using the appropriate standard procedure. Each comparison group contained 15 rats. Significance was accepted when the P value was <=0.05.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Survival in the control group C0 was 100%. In contrast, 13 (86.7%) of 15 rats in group C1 died within 36 h (P < 0.05). Bacteriological evaluation showed no infection in C0 and 100% positive blood and intra-abdominal fluid cultures in C1 (P < 0.05). Bacterial counts in the peritoneal fluid at the end of 48 h were comparable in all infected rats, averaging 9.5 x 106 ± 2.3 x 106 cfu/mL.

All intraperitoneal antibiotic treatments given immediately after challenge were better than no treatment (P < 0.05). There were significant differences in mortality between KFFKFFKFF and IKFLKFLKFL combination groups compared with polymyxin B combination group and the group that received single drugs. Survival rates were 100% for KFFKFFKFF and IKFLKFLKFL combined with levofloxacin, and 86.7% for polymyxin B and levofloxacin. In the single drug group, polymyxin B gave the highest survival rate (80%), whereas with KFFKFFKFF, IKFLKFLKFL and levofloxacin, survival was 66.7, 66.7 and 73.3%, respectively.

Qualitative blood and intra-abdominal fluid cultures showed a higher antimicrobial activity of KFFKFFKFF and IKFLKFLKFL combinations than any other treatment (P < 0.05). Similar results have been shown by quantitative bacterial cultures (P < 0.05). The results are summarized in the Table.

There were significant differences in plasma endotoxin and TNF-{alpha} levels between the control groups C0 and C1 at 60, 120 and 240 min after the challenge. In the C1 group endotoxin and TNF-{alpha} concentrations increased, with mean peak levels at 240 min post-injection (0.125 EU/mL and 25 pg/mL, respectively). All drugs and combinations produced significant reduction in plasma TNF-{alpha} levels compared with C1. However, levofloxacin significantly increased endotoxin levels compared with the control group C1. The results at 240 min post-injection are summarized in the TableGo.


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Table. Efficacy of ip polymyxins alone and combined with levofloxacin in a rat model of septic shock
 
Several compounds are currently being investigated for use in endotoxic shock; nevertheless, no treatment had showed significant benefit.1–4,7,8 Taken together our data indicate that the use of single dose intraperitoneal polymyxin can result in bacterial growth inhibition, even if the greatest efficacy was achieved only by co-administration of levofloxacin. KFFKFFKFF and IKFLKFLKFL exhibited similar antibacterial activity to polymyxin B, binding affinities for LPS and abilities to block induction of TNF. They were not completely protective themselves, indicating that they did not have high antibacterial activity alone; nevertheless, these polymyxin derivatives did not show any apparent toxicity and augmented their activity when used in combination with levofloxacin. In contrast, levofloxacin significantly increased endotoxin levels compared not only with the polymyxin-treated animals, but also with control groups, in agreement with previous observations that quinolones can promote endotoxin release.9 Definitive conclusions cannot be drawn about the anti-endotoxin effect of the agents tested because of their antibacterial activity, which reduces the number of viable bacteria. Nevertheless, our data show that the combinations as tested were efficacious in protecting rats against lethal Gram-negative septic shock. For these reasons, their use in systemic therapy in humans should be considered further.


    Notes
 
* Correspondence address. Clinica delle Malattie Infettive, c/o Ospedale Regionale, via Conca, 60020 Ancona, Italy. Tel: +39-071-5963715; Fax: +39-071-5963468; E-mail: anconacmi{at}interfree.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Wheeler, A. P. & Bernard, G. R. (1999). Treating patients with severe sepsis. New England Journal of Medicine 340, 207–14. [Free Full Text]

2 . Bone, R. C. (1993). Gram-negative sepsis: A dilemma of modern medicine. Clinical Microbiology Reviews 6, 57–68. [Abstract]

3 . Mira, J. P., Cariou, A., Grall, F., Delclaux, C., Losser, M. R., Heshmati, F. et al. (1999). Association of TNF2, a TNF{alpha} promoter polymorphism, with septic shock susceptibility and mortality. Journal of the American Medical Association 282, 561–8. [Abstract/Free Full Text]

4 . Danner, R. L., Joiner, K. A., Rubin, M., Patterson, W. H., Johnson, N., Ayers, K. M. et al. (1989). Purification, toxicity, and antiendotoxin activity of polymyxin B nonapeptide. Antimicrobial Agents and Chemotherapy 33, 1428–34. [ISI][Medline]

5 . Vaara, M. & Porro, M. (1996). Group of peptides that act synergistically with hydrophobic antibiotics against Gram-negative enteric bacteria. Antimicrobial Agents and Chemotherapy 40, 1801–5. [Abstract]

6 . Khan, A. A., Slifer, T. R. & Remington, J. S. (1998). Effect of trovafloxacin on production of cytokines by human monocytes. Antimicrobial Agents and Chemotherapy 42, 1713–7. [Abstract/Free Full Text]

7 . Abraham, E., Anzueto, A., Gutierrez, G., Tessler, S., Pedro, G. S., Wunderink, R. et al. (1998). Double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock. Lancet 351, 929–33. [ISI][Medline]

8 . Angus, D. C., Birmingham, M. C., Balk, R. A., Scannon, P. J., Collins, D., Kruse, J. A. et al. (2000). E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial. E5 Study Investigators. Journal of the American Medical Association 283, 1723–30. [Abstract/Free Full Text]

9 . Prins, J. M., van Deventer, S. J. H., Kuijper, E. J. & Spellman, P. (1994). Clinical relevance of antibiotic-induced endotoxin release. Antimicrobial Agents and Chemotherapy 38, 1211–8. [ISI][Medline]

Received 9 May 2001; returned 18 August 2001; revised 26 September 2001; accepted 12 October 2001





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