In vivo toxicity evaluation of a microsystem for polymyxin B oral administration

Abstract Summary: The aim of this study was to investigate in rats the toxicity of calcium alginate/chitosan microparticles, designed as a system to orally administer polymyxin B. Toxicity evaluation was performed after a single oral administration of PMB-loaded microparticles in comparison with unloaded microparticles and pure PMB. Introduction: The development of biodegradable microparticles with mucoadhesive properties and with a size of less than 5 μm could be very useful to promote the absorption of drugs with poor bioavailability via translocation of particles by M cells of Peyer’s patches (PP). These specialized cells staying over mucosa-associated lymphoid tissue (MALT), interspersed by enterocytes in the follicle associated epithelium (FAE), are able to transport via endocytosis a variety of microparticulate matter, macromolecules and microorganisms (bacteria, viruses, protozoae) from gut lumen to intraepithelial lymphoid cells (1). For this reason, the aim of this study was to investigate in rats the toxicity of calcium alginate/chitosan (Ca-ALG/CHT) microparticles, designed as a system to orally administer polymyxin B (PMB), antibiotic agent indicated in the treatment of serious infections caused by susceptible strains of Ps. Aeruginosa, H. influenzae, Escherichia coli, Aerobacter aerogenes, Klebsiella pneumoniae, when less potentially toxic drugs are ineffective or contraindicated. Biodegradable microparticles, prepared by spray-drying a sodium alginate solution containing PMB and subsequent reaction with Ca2+ ions and CHT, have been characterized in vitro and evaluated ex-vivo in previous studies (2). Toxicological evaluation was performed in this study in conformity with European Community regulations controlling experiments on live animals (CEE Council 89/609: Italian D.L.vo. 22-1-92 n. 116) after a single oral administration to male rats of PMB microparticles in comparison with unloaded microparticles and pure PMB. Experimental Methods: Thirty-six male rats (Wistar) from Harlan (Milan, Italy) of the same age, weighing about 200 g were housed alone in a metabolic cage with free access to water, maintained at constant room temperature (21°C). The animals were maintained at controlled temperature (21°C), humidity (50–60%) and day length (12 h) for 1 week before experiments. The toxicity study was performed after single administration to, respectively, 6 control rats and 6 assay rats for each sample: PMB water solution (60 mg/2 ml  300 mg/kg), PMB-loaded microparticle water suspension (500 mg/2 ml  60 mg/2 ml of pure PMB), unloaded microparticle water suspension (440 mg/ 2 ml). The control animals received 2 ml of distilled water. The administration was performed by oral gavage to fed/fasted rats. All treated animals were continuously monitored (mortality, behaviour, clinical signs) during the 24 h following the drug administration and urine samples were collected at 6 and 24 h. All surviving animals were killed 24 h after the treatment. After their death blood samples were collected and several organs were observed in situ to detect macroscopic abnormalities. PMB levels were determined on urine samples and serum, obtained from blood, by a microbiological agar well diffusion method using E. coli as indicator strain. Results and Discussion: PMB solution administration to fasted rats: all animals died within 30’. The only abnormal physical sign noted during post-mortem examination was the presence of an intestinal haemorrhage (Fig. 2), probably causing the death. PMB solution administration to fed rats: no animal died during the 24 h observation following the 300 mg/kg administration, but suffering signs like dyspnoea or haemorrhagic zones rounding the eyes were observed. At necropsy, all dosed rats had enlarged stomach (Fig. 1), probably causing breath problems. Therefore, after intragastric administration, PMB was lethal at a lower dose than that reported in the literature (LD50=790 mg/kg) especially in fasted animals. Fig. 1 - Comparison between stomachs excised from a control (top side) and six treated rats. Unloaded microparticles administration to fasted and fed rats: no animal died and no modification of animal behaviour and clinical signs were observed. No macroscopic abnormality in the organs examined after the animals were sacrificed was detected. These investigations thus seemed to indicate the safety of unloaded microparticles. PMB-loaded microparticles administration to fed rats: all animals were still alive over 24 h without any appreciable behavioural signs of acute toxicity. At necropsy, all dosed rats had enlarged cecae/large intestines (Fig. 2). All the other organs were normal. Considering the PMB loading efficiency and PMB weight percentage in PMB-loaded microparticles, equivalent to 300 mg/kg, PMB-loaded microparticles appear more safety than free PMB. Fig. 2 - Comparison between intestines excised from a control (top side), PMB-loaded microparticle treated rat (middle side) and free PMB treated rat (bottom side). The agar well diffusion method chosen to test antimicrobial activity in biological fluids was considered suitable to detect very low PMB concentrations in urine and serum samples. In fact, plates related to samples collected from animals treated with PMB solutions showed pronounced and measurable zone of inhibition around each well; plates related to samples from animals treated with PMB-loaded microparticles showed less pronounced inhibition zone, whereas those related to rats treated with unloaded microparticles and controls showed no zone around any well at any time. Conclusion: Intragastric administration performed by oral gavage was considered suitable because no injuries was found to be related to the way of administration. The microbiological method is relatively simple and has sufficient precision and accuracy to be used to monitor drug levels. Moreover, this study suggests that calcium alginate/chitosan microparticles were non-toxic and the intragastric administration of the microparticulate formulation determined a free PMB toxicity reduction. References: 1) T.H. Ermak, P.J. Giannasca Microparticle targeting to M cells. Adv. Drug Del. Rev., 34, 261; 1998. 2) G. Coppi, V. Iannuccelli, N. Sala, M. Bondi Alginate microparticles for polymixin B Peyer’s patches uptake. J. Microencapsulation, 21, 829; 2004.