1 Microbiology Division, 2 Pharmaceutics Department and 3 Pharmacology Department, Faculty of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Received 13 June 2004; returned 8 July 2004; revised 14 September 2004; accepted 14 September 2004
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Abstract |
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Methods: In the current work, ciprofloxacin and vancomycin were encapsulated in a cationic, anionic or neutral liposomal formulation. For prolonged circulation in serum, liposomal dispersions (<100 nm in diameter) were sonicated for different times (20, 40, 60 or 80 s), and tested for antibacterial activities.
Results and conclusions: Liposomes sonicated for 40 s gave the highest antibacterial activities in vitro. Since cationic liposomes trapped the highest percentage of antibiotics, and enhanced antibacterial activity above that of the free drugs, they were used for therapeutic trials to treat chronic staphylococcal osteomyelitis induced in rabbits. Therapeutic trials with antibiotics given intravenously revealed that, free ciprofloxacin or vancomycin given alone for 14 days was ineffective in sterilizing bone. Combination therapy with free ciprofloxacin and vancomycin for 14 days was more effective. However, this group showed renal dysfunction and severe diarrhoea, which resulted in loss of 33.3% of treated animals. Treatment with liposomal forms of either drug for 7 days was ineffective. Meanwhile, combination therapy in liposomal form for 7 days was more effective. Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group treated for 14 days with the combination of both drugs in liposomal form. Moreover, liposomal formulations showed much lower nephrotoxicity and a lower incidence of severe diarrhoea than that induced by free drugs.
Keywords: S. aureus , liposomes , ciprofloxacin , vancomycin
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Introduction |
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Liposomes have been extensively used as carriers of antimicrobial and antineoplastic drugs.9 They are usually produced from naturally occurring, biodegradable and non-toxic phospholipids.10 Liposomes have been designed to release drugs into an extracellular or intracellular compartment to reach their site of action.11 The ability of liposomes to alter drug distribution depends largely on their size and surface properties.11 Thus, liposomal encapsulation of antibiotics is a drug delivery system that helps to increase the therapeutic index of the antibiotics by increasing the concentration of the drug at the site of infection and/or reducing the toxicity.12 Organs rich in cells from the reticuloendothelial system (RES) preferentially take up liposomes, e.g. liver, spleen, lung and bone marrow.9 Targeting of liposomal antibiotic to bone marrow might achieve a high concentration of the drug in bone tissues. For extracellular bacteria, the enhanced antibacterial effect may be due to a fusion mechanism of the liposomal formulation with bacteria. Intracellularly infected phagocytic cells demonstrate that the phagocytosis of antibiotic-loaded liposomes yields therapeutic intracellular drug concentrations and consequently enhanced killing of intracellular microorganisms, such as Staphylococcus aureus, Escherichia coli, Brucella abortus and Mycobacterium avium.12
The present study was concerned with the establishment of a new strategy for antibiotic therapy against chronic staphylococcal osteomyelitic infection in rabbits. Therapeutic trials compared the ability of the liposomal form of ciprofloxacin and vancomycin alone and in combination for 7 and 14 days to sterilize the infected bones. In addition, the study was undertaken to promote more efficient liposomebacterium interaction, by testing liposomal formulations of varying characteristics regarding the size and the membrane electric charge. To the best of our knowledge, no study has yet been conducted using liposomal antibiotics for the treatment of S. aureus osteomyelitis infection.
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Materials and methods |
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A clinical strain of S. aureus isolated from a patient with chronic osteomyelitis as well as a standard strain (ATCC 29213) were used throughout this investigation. To prepare the inocula of bacterial suspensions, bacteria were grown for 18 h in trypticase soy broth (TSB), 1 mL of which was added to 10 mL of TSB and incubated for 5 h at 37°C to obtain exponential phase bacteria. Bacterial concentration was adjusted by spectrophotometry (A450) thereafter to the desired cfu/mL of phosphate buffered saline (PBS).2
Preparation of liposomes
Liposomes were prepared as previously described by Ravaoarinoro et al.13 Briefly, three liposomal compositions were formulated: lecithin (egg phosphatidyl chloine), stearylamine and cholesterol (cationic liposomes); lecithin, L-phosphatidyl-DL-glycerol and cholesterol (anionic liposomes); and lecithin and cholesterol (neutral liposomes). The molar ratio was 7:2:1, respectively, for charged liposomes, whereas it was 7:1 for neutral preparations. Lipid mixtures were dissolved in chloroform in a round-bottomed flask and dried to a thin lipid film with a rotary evaporator (Heidolph WB2000, Germany) in high vacuum at 40°C. The lipids were then hydrated by agitation in 6 mL of an aqueous solution of antibiotics at 20 mg/mL concentration for 1 h at 55°C. To manipulate the in vitro effect of liposomal particle size, lipid suspensions, submerged in an ice bath, were sonicated for 20, 40, 60 or 80 s in an ultrasonic bath (Braun-Sonic 200; Braun Instruments, Burlingame, CA, USA). Unencapsulated antibiotic was removed by centrifugation at 60 000 g for 1 h at 4°C in a Beckman ultracentrifuge (Optima-MAX-E; Beckman Coulter Inc., Palo Alto, CA, USA). The final pellets were resuspended in 6 mL of distilled water, and the liposomal preparations stored at 4°C to be used in the same week. Cholesterol, L
-phosphatidyl-DL-glycerol, egg lecithin and stearylamine were obtained from Sigma Co. (St Louis, MO, USA).
Determination of encapsulation efficiency
Encapsulation efficiency of charged or neutral liposomes was calculated as the percentage of antibiotic incorporated in liposomes relative to the initial total amount of antibiotic in solution. Antibiotic assay was performed by radial diffusion from wells cut in MuellerHinton agar (Oxoid) with a lawn of S. aureus ATCC 29213, and the antibiotic concentration was determined from the standard curve. Intra-liposomal antibiotic was calculated by two methods: first, by measuring the concentration of unentrapped antibiotic detected in supernatant after centrifugation of the liposomal preparation at 60 000 g for 1 h and subtracting this from the initial total concentration of antibiotic. The other method depended on rupturing the liposomes by adding 100 µL of 1% Triton X-100 (Sigma) in Tris/saline buffer to 400 µL of the liposomal suspension to release the entrapped antibiotic.14
Susceptibility testing
The MICs of antibiotics in free or in liposomal form were determined against the clinical as well as the standard strain (ATCC 29213) of S. aureus by an agar dilution method.15 A 4 h culture in TSB was diluted and an inoculum of 104105 cfu per spot was obtained on MuellerHinton agar plates containing different dilutions of antibiotic. After incubation for 18 h at 37°C, the plates were examined visually for the presence or absence of bacterial growth. MICs were recorded as the lowest concentrations of antibiotics that inhibited bacterial growth. All MIC determinations were performed in duplicate to ensure reproducibility of results.
Effect of liposomal size on antibacterial activity
Antibacterial activities of different liposomal formulations sonicated for different times (20, 40, 60 or 80 s) the against the standard strain of S. aureus (ATCC 29213) were determined in wells by an agar diffusion method. The experiments were performed three times and the results expressed as the mean of inhibition zone diameters in mm.
Induction and evaluation of osteomyelitis
Sixty-five New Zealand white rabbits, weighing 45 lb, were anaesthetized16 intravenously with phenobarbitone (30 mg/kg) and intramuscularly with ketamine (10 mg/kg). The left legs were shaved and disinfected with povidone iodine (Betadine). The proximal medial surface of the tibia was surgically exposed and a hole made through the cortex by a high-speed drill equipped with a 1.2 mm bit.3 Five microlitres of 50 µL/mL arachidonic acid (Sigma Co.), which acts as a sclerosing agent, was injected into the marrow cavity with a 10 µL Hamilton microsyringe (Hamilton Co., Reno, NE, USA), followed by 5 µL of S. aureus (2 x 106 cfu)1,2 and 0.1 mL of sterile saline to assure complete entry of the bacteria into the marrow. Drill holes were sealed with sterile bone wax (Ethicon Sutures Ltd, Peterborough, Ontario, Canada), and the incisions were closed with sutures. A negative control group of animals underwent the same surgical treatment as described above (including the sclerosing agent) but received 0.1 mL of sterile normal saline solution instead of S. aureus. At weekly intervals, all rabbits were weighed. Osteomyelitis was evaluated by palpation, sinus formation and pus discharge, and by radiology. Two observers independently examined X-rays of the infected tibiae for evidence of osteomyelitis 2 weeks after injection and at weekly intervals thereafter. The severity of disease was determined according to the scale of 03 described by Norden et al.17
The in vivo study adhered to the principles and ethical standards of laboratory animal care that are approved by the Research Center at the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
Conduct of therapeutic trials
Since radiological changes in chronic osteomyelitis were present at day 14, treatment at this time was considered to represent therapy for chronic osteomyelitis.7 Therapy was instituted 14 days after infection and given for 14 days. The rabbits still alive were divided into 10 groups (six in each); each group received one of the following regimens: (i) no antibiotic therapy; (ii) twice daily injection of ciprofloxacin (10 mg/kg); (iii) twice daily injection of vancomycin (15 mg/kg); (iv) twice daily injection of vancomycin and ciprofloxacin at doses mentioned above; (v) twice daily injection of ciprofloxacin (10 mg/kg) in liposomal form; (vi) twice daily injection of vancomycin (15 mg/kg) in liposomal form; (vii) twice daily injection of ciprofloxacin and vancomycin in liposomal form at the doses mentioned above. In addition, the three liposomal forms of the antibiotics were tried for 7 days. All injections were given intravenously. All rabbits were killed at the end of the experiments after blood samples had been taken. Bone marrow specimens were weighed, suspended in 2 mL of sterile PBS (pH 7.4), and agitated by vigorous vortexing to dislodge the organisms attached; if any.18 Blood samples and bone marrow specimens were cultured on MuellerHinton agar plates for bacterial growth.
Renal function and antibiotic therapy
The serum urea and creatinine levels were measured after a 2 week course of antibiotic therapy.
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Results |
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The percentage of ciprofloxacin and vancomycin trapped in cationic, anionic and neutral liposomes is shown in Table 1. A greater percentage of each antibiotic was trapped within charged liposomes than within neutral liposomes.
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MICs of antibiotic-loaded liposomes compared with free antibiotics against S. aureus strains are shown in Table 2. With the exception of cationic liposomal forms, other formulations yielded MICs equal to or higher than those obtained with the free drug. MICs obtained with cationic liposomes matched those obtained with the free drug or were one or two dilutions lower.
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The results shown in Table 3 indicate that positively charged liposomal preparations of antibiotics sonicated for 40 s gave the highest antibacterial activity against the standard strain (S. aureus ATCC 29213) by agar diffusion.
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Injection of arachidonic acid alone produced no gross or radiological evidence of osteomyelitis. Only rabbits injected with arachidonic acid and the clinical isolate of S. aureus developed radiological evidence of osteomyelitis. Some widening and distortion of normal bone architecture can be seen in Figure 1(a) compared with a control tibia shown in Figure 1(b). Out of 65 rabbits injected with 2 x 106 S. aureus, five rabbits (7.7%) died within 14 days of injection. Cultures of bone from all five dead rabbits yielded the injected S. aureus.
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The serum urea and creatinine levels were measured 2 weeks after therapy. As shown in Table 5, rabbits that received a combination of free ciprofloxacin and vancomycin therapy for 14 days often developed severe diarrhoea and renal dysfunction. This situation resulted from the mean serum urea and creatinine of this group, which were shown to be significantly different from the control group and from the group that received liposomal combination therapy.
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Discussion |
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Previous studies with a rabbit model of staphylococcal osteomyelitis have shown that the use of single antibiotics in free form, including ciprofloxacin and vancomycin, or in combination with another antibiotic, is only moderately effective in sterilizing infected rabbit bones.1722 The animal model poses a difficult challenge for any antimicrobial agents because to be considered successful in clinical trials it requires sterilization of the infected bone.23
The aim of this study was to develop a new regimen for treating a rabbit model of chronic staphylococcal osteomyelitis by antibiotics encapsulated in liposomes. Liposomes offer a safe and convenient means to control the rate and location by which drugs are delivered within the body. Liposomal size and surface charges can be manipulated and optimized for each therapeutic application.11 A major problem in the targeting of intravenously injected liposomes to the mononuclear phagocytes of the bone marrow is their rapid and efficient removal by the RES of Kupffer cells of the liver and macrophages of the spleen.24 Thus one of our goals in this study was the reduction of RES uptake of liposomes by liver and spleen by means of surface charge and size modification, and hence a substantially prolonged blood circulation time. The results presented in Table 1 demonstrate that ciprofloxacin and vancomycin could be incorporated within anionic, cationic and neutral liposomes. The highest entrapment that was observed with cationic followed by anionic liposomes supports the view that the presence of charged lipid increases the aqueous space with liposomes. The encapsulation efficiency of both drugs in cationic liposomes was 9% higher than that of anionic liposomes (Table 1). Moreover, the in vitro activity of anionic and neutral liposomes containing ciprofloxacin or vancomycin was reduced against the clinical isolate of S. aureus compared with the free forms of the drugs (Table 2). However, the cationic liposomal formulations of these drugs enhanced their in vitro activity against the clinical isolate, and yielded MICs equal or lower than those obtained with the free drug (Table 2). Such enhancement could be explained on the basis that, in cationic liposomes, the external molecules supply a positive charge to vesicles. Thus, interaction of negatively charged bacterial cell surfaces with cationic liposomes might take place.25 In addition, positively charged liposomes are cleared less rapidly from the circulation than negatively charged liposomes.26
To obtain a uniform and reproducible liposomal particle size between 50100 nm, the method described by Omri et al.15 was followed. Liposomes <100 nm in diameter are less efficiently taken up by the RES and may have a considerably longer mean residence time in plasma,11 which would be an advantage for treatment of osteomyelitis. Liposomal size produced by sonication for 40 s was the most efficient preparation in vitro (Table 3). Based on the in vitro studies, cationic liposomes sonicated for 40 s appear to be an interesting carrier and should be considered for further evaluation in vivo.
In the present study, all untreated rabbits had positive cultures when killed at the end of experiments. Treatment with either free ciprofloxacin or vancomycin for 14 days was ineffective in sterilizing the bones of rabbits with staphylococcal osteomyelitis. The combination of both drugs in free form was more effective in sterilizing infected bones than either agent alone. Despite some cures seen (33.3%) in the experimental osteomyelitis group treated with a combination of free drugs (Table 4), these cannot be taken to imply a recommendation for treatment of chronic osteomyelitis with free antibiotics alone.7 Adjuvant therapy such as surgery is recommended.27 Administration of either ciprofloxacin or vancomycin alone or in combination in liposomal form for 7 days was proposed in this study to determine the usefulness of short-term unconventional therapy. A short course would reduce the cost, toxicity and the inconvenience to patients as liposomal preparations are administered intravenously. These trials showed monotherapy was insufficient to sterilize bone (Table 4). Administration of a combination of both drugs in liposomal form for 7 days, or either drug alone in liposomal form for 14 days was more effective, but did not sterilize all bone tissues (Table 4). A combination of ciprofloxacin and vancomycin administered in liposomal form for 14 days sterilized the bones of all treated animals. These results are significantly better than those described in other studies which used single or combination therapy.1722 One of the possible explanations for the relative success of the combination of both drugs in liposomal form might be their ability to penetrate into macrophages and RES in concentrations high enough to cause sterilization of the bones. Moreover, the cationic liposomal forms prolonged the circulation time and decreased the uptake of drugs in liposomal form by macrophages in the liver or spleen. Ciprofloxacin, in free form, is generally well tolerated with relatively low occurrence of adverse reactions.28 Among the adverse reactions induced by free vancomycin therapy are: severe watery diarrhoea and renal impairment manifested by increasing serum urea and creatinine, especially in patients exposed to a high dose of vancomycin.29 Because of renal dysfunction, the dose and duration of vancomycin administration are limited.29 Protection of kidneys might permit the administration of high doses of vancomycin for longer periods. The study revealed that, combination therapy in liposomal form is significantly better than combination therapy in free form in reducing drug toxicity. The data shown in Table 5 revealed that liposomal encapsulation of drugs has a profound effect on reduction of nephrotoxicity associated with these formulations. This significant reduction appears to correlate with decreased renal drug uptake11 and modification in drug distribution.9
In conclusion, it seems likely that a combination therapy of ciprofloxacin and vancomycin in liposomal form for 2 weeks will be a valuable addition to therapeutic regimens for chronic osteomyelitis caused by S. aureus and should be considered in future for clinical trials in human osteomyelitis.
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Acknowledgements |
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Footnotes |
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References |
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2 . Power, M. E., Olson, M. E., Domingue, P. A. et al. (1990). A rat model of S. aureus chronic osteomyelitis that provides a suitable system for studying the human infection. Journal of Medical Microbiology 33, 18998.[Abstract]
3 . Hienz, S. A., Sakamoto, H., Flock, J. I. et al. (1995). Development and characterization of a new model of hematogenous osteomyelitis in the rat. Journal of Infectious Diseases 171, 12306.[ISI][Medline]
4 . Mayberry-Carson, K. J., Tober-Meyer, B., Smith, J. K. et al. (1984). Bacterial adherence and glycocalyx formation in osteomyelitis experimentally induced with S. aureus. Infection and Immunity 43, 82533.[ISI][Medline]
5 . Fresta, M., Spadaro, A., Cerniglia, G. et al. (1995). Intracellular accumulation of ofloxacin-loaded liposomes in human synovial fibroblasts. Antimicrobial Agents and Chemotherapy 39, 13725.[Abstract]
6 . Widmer, A. F. (2001). New developments in diagnosis and treatment of infection in orthopedic implants. Clinical Infectious Diseases 33, S94106.[CrossRef][ISI][Medline]
7 . Norden, C. W. & Kennedy, E. (1971). Experimental osteomyelitis. II. Therapeutic trials and measurement of antibiotic levels in bone. Journal of Infectious Diseases 124, 56571.[ISI][Medline]
8 . Norden, C. W. & Kennedy, E. (1970). Experimental osteomyelitis. I. A description of the model. Journal of Infectious Diseases 122, 4108.[ISI][Medline]
9 . Berestein, G. L. (1987). Liposomes as carriers of antimicrobial agents. Antimicrobial Agents and Chemotherapy 31, 6758.[ISI][Medline]
10
.
Furneri, P. M., Fresta, M., Puglisi, G. et al. (2000). Ofloxacin-loaded liposomes: in vitro activity and drug accumulation in bacteria. Antimicrobial Agents and Chemotherapy 44, 245864.
11 . Fielding, R. M. (1991). Liposomal drug delivery: advantages and limitations from a clinical pharmacokinetics and therapeutic perspective. Clinical Pharmacokinetics 21, 15564.[ISI][Medline]
12
.
Schiffelers, R., Storm, G. & Woudenberg, I. B. (2001). Liposome-encapsulated aminoglycosides in pre-clinical and clinical studies. Journal of Antimicrobial Chemotherapy 48, 33344.
13 . Ravaoarinoro, M., Toma, E., Agbaba, O. et al. (1993). Efficient entrapment of amikacin and teicoplanin in liposomes. Journal of Drug Targeting 1, 1915.[Medline]
14 . Morgan, J. R. & Williams, K. E. (1980). Preparation and properties of iposome-associated gentamicin. Antimicrobial Agents and Chemotherapy 17, 5448.[ISI][Medline]
15 . Omri, A., Ravaoarinoro, M. & Poisson, M. (1995). Incorporation, release and in vitro antibacterial activity of liposomal aminoglycosides against P. aeruginosa. Journal of Antimicrobial Chemotherapy 36, 6319.[Abstract]
16 . Illero, J. C., Gonzalez, G. A., Silvan, G. et al. (2000). The effects of different anaesthetic treatments on the adreno-cortical functions and glucose levels in NZW rabbits. Journal of Physiology and Biochemistry 56, 32936.[ISI][Medline]
17 . Norden, C. W., Shinner, E. & Niederriter, K. (1986). Clindamycin treatment of experimental chronic osteomyelitis due to S. aureus. Journal of Infectious Diseases 153, 9569.[ISI][Medline]
18 . Norden, C. W. & Keleti, E. (1980). Treatment of experimental staphylococcal osteomyelitis with rifampin and trimethoprim, alone and in combination. Antimicrobial Agents and Chemotherapy 17, 5914.[ISI][Medline]
19 . Norden, C. W. & Shinners, E. (1985). Ciprofloxacin as therapy for experimental osteomyelitis caused by Pseudomonas aeruginosa. Journal of Infectious Diseases 151, 2914.[ISI][Medline]
20 . Norden, C. W. (1983). Experimental chronic staphylococcal osteomyelitis in rabbits: treatment with rifampin alone and in combination with other antimicrobial agents. Review of Infectious Diseases 5, Suppl. 3, S4914.[ISI][Medline]
21 . Norden, C. W. & Shaffer, M. (1983). Treatment of experimental chronic osteomyelitis due to S. aureus with vancomycin and rifampin. Journal of Infectious Diseases 147, 3527.[ISI][Medline]
22 . Norden, C. W. (1975). Experimental osteomyelitis. IV. Therapeutic trials with rifampin alone and in combination with gentamicin, sisomicin, and cephalothin. Journal of Infectious Diseases 132, 4939.[ISI][Medline]
23 . Norden, C. W. & Budinsky, A. (1990). Treatment of experimental chronic osteomyelitis due to Staphylococcus aureus with ampicillin/sulbactam. Journal of Infectious Diseases 161, 523.[ISI][Medline]
24 . Moghimi, S. M., Porter, C. J. H., Illum, L. et al. (1991). The effect of poloxamer-407 on liposome stability and targeting to bone marrow: comparison with polystyrene microspheres. International Journal of Pharmaceutics 68, 1216.[CrossRef][ISI]
25 . Trafny, E. A., Stepinska, M., Antos, M. et al. (1995). Effects of free and liposome-encapsulated antibiotics on adherence of Pseudomonas aeruginosa to collagen type I. Antimicrobial Agents and Chemotherapy 39, 26459.[Abstract]
26 . Tyrrell, D. A., Heath, T. D., Colley, C. M. et al. (1976). New aspects of liposomes. Biochimica et Biophysica Acta 457, 259302.[ISI][Medline]
27 . Norden, C. W. & Keleti, E. (1980). Experimental osteomyelitis caused by Pseudomonas aeruginosa. Journal of Infectious Diseases 141, 715.[ISI][Medline]
28 . Sprandel, K. A. & Rodvold, K. A. (2003). Safety and tolerability of fluoroquinolones. Clinical Cornerstone 5, Suppl. 3, S2936.
29 . Nishino, Y., Takemura, S., Minamiyama, Y. et al. (2002). Inhibition of vancomycin-induced nephrotoxicity by targeting superoxide dismutase to renal proximal tubule cells in the rat. Redox Report 7, 3179.[CrossRef][ISI][Medline]
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