1 Institute of Infectious Diseases and Public Health, Università Politecnica delle Marche, Ancona; 2 Department of General Surgery, I.N.R.C.A.I.R.R.C.S., Università Politecnica delle Marche, Ancona; 3 Biotechnology Centre, Research Department, I.N.R.C.A.I.R.R.C.S., Ancona, Italy; 4 Faculty of Pharmacy, Medical University of Gdansk, Gdansk, Poland
Received 1 April 2004; returned 28 April 2004; revised 28 June 2004; accepted 1 July 2004
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Abstract |
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Methods: The in vitro antimicrobial activity of MSI-78 was investigated against the commercially available quality control strain Escherichia coli ATCC 25922. In addition, three rat models of septic shock were investigated: (i) rats were injected intraperitoneally with 1 mg Escherichia coli 0111:B4 LPS; (ii) rats were given an intraperitoneal injection of 2 x 1010 cfu of Escherichia coli ATCC 25922; (iii) intra-abdominal sepsis was induced via caecal ligation and puncture. All animals were randomized to receive after 360 min intravenously isotonic sodium chloride solution, 1 mg/kg MSI-78, or 60 mg/kg piperacillin. Main outcome measures were: abdominal exudate and plasma bacterial growth, plasma endotoxin and tumour necrosis factor (TNF-
) concentrations, and lethality.
Results: Our in vitro data showed that MSI-78 possesses a strong activity against Escherichia coli. The in vivo studies showed that all compounds reduced the lethality when compared to controls. MSI-78 showed a slightly higher antimicrobial activity than piperacillin and achieved a substantial decrease in endotoxin and TNF- plasma concentrations than the ß-lactam.
Conclusions: Because of its strong double anti-endotoxin and antimicrobial activities MSI-78 could be an interesting compound for Gram-negative septic shock treatment.
Keywords: septic shock , endotoxin , peptides
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Introduction |
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Among these animal-derived antimicrobial agents are the magainins, discovered in the skin of the African clawed frog Xenopus laevis more than 15 years ago.21 Similar to other polycationic peptides, which are -helical ionophores, they possess two important activities: a broad antimicrobial spectrum and anti-endotoxin activity. Through a series of amino acid deletions and substitution, MSI-78 was created. It is a 22 residue magainin analogue that showed an enhanced in vitro and in vivo potency relative to that of magainin 2 against both Gram-positive and Gram-negative bacteria.2224
The present experimental study was designed to investigate the in vivo efficacy of MSI-78 in three rat models of septic shock.
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Materials and methods |
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Adult male Wistar rats weighing 200250 g were used for all the experiments. All animals were housed in individual cages under constant temperature (22°C) and humidity with a 12-h light/dark cycle, and had access to chow and water ad libitum throughout the study. The study was approved by the animal research ethics committee of the I.N.R.C.A.I.R.R.C.S., Polytechnic University of Marche, Ancona, Italy.
Organisms and reagents
The commercially available quality control strain of Escherichia coli ATCC 25922 was used. Endotoxin (Escherichia coli serotype 0111:B4; SigmaAldrich S.r.l., Milan, Italy) was prepared in sterile saline, and stored in aliquots at 80°C for short periods.
Agents
MSI-78 (Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys-NH2) was synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry according to the following procedure: (i) 2 and 20 min deprotection steps using 20% piperidine in dimethylformamide (DMF) in the presence of 1% Triton; (ii) the coupling reactions were carried out with the protected amino acid diluted in DMF in the presence of 1% Triton using diisopropylcarbodiimide (DIC) as the coupling reagent in the presence of 1-hydroxybenzotriazole (HOBt) for 2 h. The completeness of each coupling reaction was monitored by the chloranil test.25,26 The protected peptidyl resin was treated with the mixture: 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (TIS) for 2 h. After cleavage, the solid support was removed by filtration, and the filtrate was concentrated under reduced pressure. The cleaved peptide was precipitated with diethyl ether and lyophilized. Pexiganan was purified by high-pressure liquid chromatography (HPLC) on a Knauer K501 two-pump system. The resulting fractions with purity greater than 9798% were tested by HPLC. The peptide was analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF).
Piperacillin (SigmaAldrich) powder was diluted in accordance with manufacturer's recommendations. Solutions were made fresh on the day of assay.
Susceptibility testing
Susceptibility testing was carried out by microbroth dilution method according to the procedures outlined by the National Committee for Clinical Laboratory Standards.27 However, since cationic peptides bind polystyrene, polypropylene 96-well plates (SigmaAldrich) were substituted for polystyrene plates. The MIC was taken as the lowest antibiotic concentration at which observable growth was inhibited. Experiments were carried out in triplicate.
Experimental design
Septic shock was induced under three different experimental conditions: (i) intraperitoneal administration of LPS, (ii) E. coli induced peritonitis, (iii) caecal ligation and puncture.
For each animal model, toxicity was evaluated on the basis of the presence of any drug-related adverse effects, i.e. local signs of inflammation, anorexia, weight loss, vomiting, diarrhoea, fever and behavioural alterations. In particular, to evaluate the physiological effects of MSI-78, rectal temperature, pulse, blood pressure, breathing rate and oxygenation were monitored in a supplementary MS-78-treated group without infection or LPS.
Serum antibiotic concentration measurements and kinetics
Preventive experiments were carried out to measure serum MSI-78 and piperacillin levels in uninfected animals. Blood samples were obtained from the tail vein of 12 rats (six rats for each agent) 0.50, 1, 2 and 8 h after a single intravenous dose of MSI-78 (1 mg/kg) and piperacillin (60 mg/kg). Drug levels were measured by bioassay: a spore suspension of Bacillus subtilis ATCC 6633 suspended in tryptic soy agar was used. The plates were read after incubation at 30°C for 18 h.
Evaluation of treatment
On the basis of the type of experiment, at the end of the study, the rate of blood culture positivity, the quantities of bacteria in the intra-abdominal fluid, the rate of lethality, and plasma endotoxin and tumour necrosis factor (TNF-
) levels were evaluated. The animals were monitored for the subsequent 72 h. In models (ii) and (iii), the presence of sepsis 6 h after bacterial challenge was defined by analogy to the criteria applied for humans. Each animal was considered to be septic if it satisfied at least two of the following criteria: a) increased pulse rate; b) rectal temperature above 38°C or below 36°C; c) increased breathing rate.
The surviving animals [(models (ii) and (iii)] were killed with chloroform, and blood samples for culture were obtained by aseptic percutaneous transthoracic cardiac puncture. In addition, to perform quantitative evaluations of the bacteria in the intra-abdominal fluid, 10 mL of sterile saline was injected intraperitoneally, samples of the peritoneal lavage fluid were serially diluted, and a 0.1 mL volume of each dilution was spread onto blood agar plates. The limit of detection was 1 log10 cfu/mL. The plates were incubated both in air and under anaerobic conditions at 35°C for 48 h.
For blood cultures and determination of endotoxin and TNF- in plasma, 0.2 mL blood samples were collected from a tail vein 0, 2, 6 and 12 h after injection into a sterile syringe and transferred to tubes containing ethylenediaminetetraacetic acid tripotassium salt (EDTA-K3).
Biochemical assays
Endotoxin concentrations were measured by a Limulus amoebocyte lysate test. Plasma samples were serially two-fold diluted with sterile endotoxin-free water and were heat-treated for 5 min in a water bath at 75°C to destroy inhibitors that can interfere with the activation. 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- levels were measured with a solid phase sandwich enzyme-linked immunosorbent assay. The standards and samples were incubated with a TNF-
antibody coating 96-well microtitre plate. The wells were washed with buffer and then incubated with biotinylated anti-TNF-
antibody conjugated to streptavidin-peroxidase. This was washed away and colour was developed in the presence of chromogen (tetramethylbenzidine) substrate. The intensity of the colour was measured in a MR 700 Microplate Reader (Dynatech Laboratories, Guernsey, UK) by reading the absorbance at 450 nm. The results for the samples were compared with a standard curve to determine the amount of TNF-
present. All samples were run in duplicate. The lower limit of sensitivity for TNF-
by this assay was 0.05 ng/mL.
Statistical analysis
MIC values are presented as the average values obtained in triplicates on three independent measurements. Mortality rates between groups were compared using Fisher's exact test. Qualitative results for blood cultures were analysed by the 2 test with Yates' correction or Fisher's exact test, depending on the sample size. Quantitative evaluations of the bacteria in the intra-abdominal fluid cultures were presented as means ± standard error of the mean (S.E.M.). Statistical comparisons between groups were made by analysis of variance. Multiple comparisons were carried out by Tukey's test. As a result of the presence of several values below the lower limit of sensitivity, plasma endotoxin levels were compared between groups by KruskalWallis non-parametric test, adjusted for ties, followed by Dunn's procedure for multiple comparisons. Each comparison group contained 30 rats. Significance was accepted when the P value was
0.05.
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Results |
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According to the broth-microdilution method, E. coli ATCC 25922 was similarly susceptible to MSI-78 (MIC 0.5 mg/L) and to the control agent piperacillin (MIC 1 mg/L).
In vivo studies
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Finally, none of the treated animals had clinical evidence of drug-related adverse effects and no changes in physiological parameters were observed in the supplementary 1 mg/kg MSI-78-treated group without infection.
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Discussion |
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Like the other polycationic peptides, MSI-78 demonstrated broad spectrum in vitro antimicrobial activity against most of the common pathogens responsible for intra-abdominal sepsis. Interestingly, since it acts directly on the anionic phospholipid of the bacterial cell membrane and not on membrane receptors, the development of resistance is theoretically less likely to occur.23,24
A growing body of research has been devoted to studying animal models of septic shock and a single dose of endotoxin or a single inoculum of one Gram-negative species has been the most used for screening of anti-endotoxin and antimicrobial drugs.12,3234 On the other hand, Gram-positive or mixed Gram-negative and Gram-positive infections are also common in humans. Therefore, in this study, we used not only the animal models of intraperitoneal administration of LPS or E. coli inoculum but also the model of caecal ligation and puncture to resemble the clinical condition of a bowel perforation and mixed bacterial infection. Moreover, in our experiments, we administered the drugs 360 min after the bacterial challenge or the surgical procedure to mimic the clinical situation where an interval between the onset of sepsis and the initiation of therapy is present. Piperacillin was used as control agent. It acts against most of the pathogens that can cause intra-abdominal infections including anaerobes, Gram-positive cocci and Enterobacteriaceae.35
Data analysis for our three experiments did not show any important different impacts on parameter evaluation. In fact, it was evident that the efficacies of the compounds were not affected by the animal models used and that these were retained regardless of the system used. Overall, the administration of all agents yielded a lower rate of mortality than was seen in saline-treated controls. MSI-78 was effective against all parameters considered, regardless of the animal model utilized. Importantly, single intraperitoneal or intravenous doses of MSI-78 produced a significant reduction in the TNF- plasma levels, compared with both control and piperacillin-treated groups. Our data confirm that piperacillin, like other clinically used antibiotics, can stimulate the release of endotoxin and then increase the rates of occurrence of symptoms and life-threatening complications.3638 However, in models (ii) and (iii), MSI-78 showed the lowest lethality rate but no significant differences were observed in the rate of lethality among MSI-78- and piperacillin-treated groups. It is important to point out that the greatest significant differences in defining the efficacy of the compounds were observed in their capacity to inhibit the release of LPS, while no statistically significant differences were noted in their antimicrobial activity. The relevance of this pivotal role of antimicrobial activity has been clearly shown by the third experimental model where various Gram-negative and Gram-positive bacterial species were involved in the pathogenesis of the sepsis. In fact, lethality data demonstrated that the strong activity and broad antimicrobial spectrum of MSI-78 prevailed against the other agents. Although the extrapolation of results from animal models to human pathology should be regarded with caution, our results highlight the high therapeutic potential of MSI-78. As a result of its strong antimicrobial activity and its ability to neutralize the biological effects of endotoxin, MSI-78 may play a crucial role in the treatment of intra-abdominal sepsis.
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Acknowledgements |
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Footnotes |
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