a Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia and b Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Università degli Studi di Pisa, Pisa, Italy; c Farmigea S.p.A., Pisa, Italy
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Abstract |
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Introduction |
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Recently, a novel mucoadhesive polymer, consisting of a natural polysaccharide extracted from tamarind seeds and referred to as tamarind-gum polysaccharide (TGP), has been described as a viscosity enhancer with mucomimetic activity.4 Purified TGP is a high-molecular-weight, non-ionic, neutral, branched polysaccharide consisting of a cellulose-like backbone that carries xylose and galactoxylose substituents,4 chemical residues similar to that of mucin MUC-1 and episialin.5 Several properties make TGP an attractive candidate for use as a vehicle for ophthalmic medicaments: (i) it exhibits optimal performance as a tear fluid substitute;6 (ii) it prevents alterations of the corneal surface known as keratoconjunctivitis sicca;6 (iii) it reduces the in vitro toxicity exerted by ofloxacin, rufloxacin, timolol and merthiolate on human conjunctival cells;7 (iv) it retains its properties when autoclaved;4 and (v) in contrast to other mucoadhesive polymers,3 it maintains its characteristics at neutral pH.4 The present study was thus designed to ascertain whether TGP could enhance the trans-corneal disposition of hydrophilic and hydrophobic antimicrobial drugs, such as gentamicin and ofloxacin, when administered topically to healthy rabbits by an ocular drop regimen.
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Materials and methods |
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Sterile water solutions of gentamicin or ofloxacin contained mannitol 50 mg/mL (Sigma, St Louis, MO, USA), sodium merthiolate 0.02 mg/mL (CFM Oskar Tropitzsch, Marktredwitz, Germany), gentamicin sulphate 5 mg/mL (corresponding to 3 mg/mL gentamicin) (Sigma) or ofloxacin 3 mg/mL (Sigma). The viscosified solutions of antimicrobials contained TGP (Farmigea S.p.A., Pisa, Italy) at a concentration of 20 mg/mL. Farmigea S.p.A. kindly provided all the antibiotic formulations.
Experimental design
A total of 40 New Zealand male rabbits (22.5 kg) were used throughout the experiments and were subjected to an ocular drop regimen according to a treatment schedule described previously.1 In particular, treatment consisted of the instillation of gentamicin or ofloxacin (right eye) and the corresponding TGP formulations (left eye) into the lower conjunctival sac every 30 min for 6 h. Fifty microlitres of drug solution, corresponding to 150 µg gentamicin or ofloxacin, was applied with or without 1 mg TGP at each instillation. At fixed time intervals after the last administration (30, 60, 90, 120, 150 and 180 min), animals (five rabbits for each formulation and time analysed) were killed by injecting an overdose of ethyl urethane into the marginal ear vein. Examination of rabbit eyes during and at the end of treatment with antibiotics alone or with antibiotics viscosified with TGP did not reveal any signs of toxicity, including crusting, hyperaemia or lid swelling. Aqueous humour was aspirated by anterior chamber paracentesis using a 26-gauge needle. Corneas were taken from the animals killed 60 min after the last administration. For this purpose, eyes were rapidly enucleated, trimmed of all adventitial tissue and rinsed with sterile saline. Following excision, corneas were weighed, suspended (25 mg/mL) in phosphate-buffered saline (PBS) pH 7.2, homogenized, and centrifuged as described previously.8 Samples of aqueous fluid and corneal tissue were analysed immediately or stored at 30°C and examined within 1 month. The study was approved by the Ethics Committee of Pisa University Hospital.
Drug concentration assay
The antibiotic concentration in corneal and aqueous samples was measured by agar-diffusion bioassay using Bacillus subtilis ATCC 6633 as the indicator organism.1 B. subtilis spores were prepared in 0.1 M PBS pH 8.0 and stored at 4°C as sterile suspensions. For assays, 5 x 105 spores/mL were incorporated into molten Antibiotic Medium No. 5 or No. 2 (Difco, Detroit, MI, USA) in order to measure gentamicin and ofloxacin concentrations, respectively. The assay plates (90 mm diameter) were pierced with six holes (7 mm diameter) which were filled with 100 µL of two-fold dilutions of aqueous or corneal samples. After incubation at 37°C for 24 h, the diameter of growth inhibition halos was taken as the measure of drug concentration, by reference to calibration curves constructed by adding known amounts of gentamicin (from 0.01 to 5 mg/L) or ofloxacin (from 0.05 to 10.0 mg/L) to pooled aqueous humour or homogenized corneas taken from untreated animals. Assays were shown to be linear in the range 0.023.5 mg/L for gentamicin and 0.210.0 mg/L for ofloxacin. Each determination was performed in triplicate and the mean value was calculated; inter-assay and inter-day variations corresponding to one dilution step were observed in no more than 3% of the experiments. No growth inhibition zones were detected when wells were filled with 100 µL of TGP, either alone or in combination with the excipients (mannitol and sodium merthiolate).
Statistical and pharmacokinetic analysis
Results were expressed as mean ± standard deviation (s.d.). Statistical analysis was performed using the two-tailed Student's t-test. Pharmacokinetic analysis was carried out by fitting mean drug concentrations versus time data sets according to a one-compartment model. For this purpose a non-linear least-squares regression analysis was used (APO2PR software, MediWare, Groeningen, The Netherlands). Pharmacokinetic calculations were performed following standard methods.9 The peak concentration (Cmax) and the time to reach peak concentration (Tmax) values were identified from the inspection of drug concentrationtime plots. The t1/2 for each elimination exponential (t1/2ß) was obtained from the equation t1/2ß = 0.693/Ke, where Ke (elimination constant) is the slope of the exponential. The area under the concentrationtime curve (AUC) was calculated by the trapezoidal method for the area from the time 30 min (t30) to the time of the last quantified drug concentration (t180). The mean residence time (MRT) was also estimated.
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Results and discussion |
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The potential utility of TGP as an ophthalmic delivery system for the prevention of ocular infections may be argued considering the known MIC values of gentamicin and ofloxacin for bacteria frequently causing such infections. Indeed, in contrast to ofloxacin alone, the use of TGPofloxacin in healthy rabbits led to the attainment of aqueous antibiotic concentrations (Figure, part a) consistently higher than the MIC values reported for Pseudomonas aeruginosa (MIC90 = 3.1 mg/L),10 an organism often associated with severe intraocular infections. Similar conclusions can be drawn for gentamicin (Figure
, part b), since in the absence of TGP, this antibiotic usually achieves aqueous drug levels lower than the MIC values (0.0622 mg/L) reported frequently for common ocular pathogens including Staphylococcus aureus, Staphylococcus epidermidis and several P. aeruginosa clinical isolates.1,3 The remarkably high levels of ofloxacin and gentamicin obtained in the cornea after administration with TGP also indicate that this mucoadhesive polymer may be useful in the topical treatment of bacterial keratitis. Indeed, the concentrations of both drugs in the cornea greatly exceeded the MICs reported for most Pseudomonas, Streptococcus and Staphylococcus spp. strains frequently isolated from cases of keratoconjunctivitis.10
The overall results obtained during this investigation suggest that the tamarind seed polysaccharide can be used successfully as an ophthalmic delivery system for antimicrobial drugs and possibly for other medicaments in the topical treatment of eye diseases.
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Acknowledgments |
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Notes |
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References |
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2 . Davies, N. M., Farr, S. J., Hadgraft, J. & Kellaway, I. W. (1991). Evaluation of mucoadhesive polymers in ocular drug delivery. I: Viscous solutions. Pharmacological Research 8, 103943.
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4 . Saettone, M. F., Burgalassi, S., Boldrini, E., Bianchini, P. & Luciani, G. (1997). Ophthalmic solutions viscosified with tamarind seed polysaccharide. International patent application PCT/IT97/00026.
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8 . Engel, L. S., Callegan, M. C., Hill, J. M., Folkens, A. T., Shimomura, Y. & O'Callaghan, R. J. (1996). The effectiveness of two ciprofloxacin formulations for experimental Pseudomonas and Staphylococcus keratitis. Japanese Journal of Ophthalmology 40, 2129.[ISI][Medline]
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10 . Osato, M. S., Jensen, H. G., Trousdale, M. D., Bosso, J. A., Borrmann, L. R., Frank, J. et al. (1989). The comparative in vitro activity of ofloxacin and selected ophthalmic antimicrobial agents against ocular bacterial isolates. American Journal of Ophthalmology 108, 3806.[ISI][Medline]
Received 21 February 2000; returned 23 May 2000; revised 9 June 2000; accepted 10 July 2000