The primary organisms involved in ocular infections are Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus species, and Haemophilus influenzae.1 Conventional medical treatment of these infections typically include vancomycin and cefazolin, both of which have poor aqueous penetration and are not available in a commercial form for topical administration. In contrast, fluoroquinolones such as ofloxacin, lomefloxacin and ciprofloxacin are commercially available for topical administration. Fluoroquinolones are highly soluble and have a broad spectrum of activity. They offer the potential for broad coverage against infection if sufficient quantities of antibiotic penetrate the eye to achieve high levels in aqueous humour.2 These properties make fluoroquinolones attractive enough for treatment of ocular infections.
Ciprofloxacin eye drops are indicated for the treatment of corneal ulcers and superficial infections of the eye and adnexa caused by susceptible organisms.345 The most frequently noted unwanted ocular event associated with ciprofloxacin therapy is a white crystalline precipitate, commonly located in the superficial portion of the corneal area of inflammation.5 This problem was noted in 16.6% of the patients5 by some authors who also noted that though the precipitate resolved fast and did not appear to cause any scarring, it caused a temporary decrease in vision in some. Precipitation of ciprofloxacin occurs due to a change in pH of the eye drop as it mixes with the tear film.6
Lomefloxacin is indicated for the treatment of acute bacterial conjunctivitis.78 Lomefloxacin is well tolerated, with no serious systemic or local adverse drug reactions. Lomefloxacin eye drops, used in the form of a loading dose one drop at 5 minute interval up to 2 hours followed by twice daily instillation, proved as effective, safe and well tolerated as five established standard treatments used twice, four times or five times daily.9
Ofloxacin is indicated for the topical treatment of external ocular infections, such as conjunctivitis and keratoconjunctivitis, caused by sensitive organisms in adults as well as children.101112 The efficacy and toxicity of ofloxacin in treating bacterial keratitis was found to be equivalent to that of ciprofloxacin solution.13
There is a significant increase in the rate of in vitro resistance of Staphylococcus aureus to ciprofloxacin and ofloxacin, from less than 6 percent to 35 percent over a period of 4 years.14 Garg et al15 reported that of 141 culture-proven cases of Pseudomonas keratitis, 22 cases were caused by isolates resistant to ciprofloxacin.In a situation where resistance is increasing with established antibiotics, newer fluoroquinolones, such as enoxacin, fleroxacin, levofloxacin, perfloxacin and sparfloxacin, are evaluated extensively as ocular therapeutic agents.16
Amongst the newer fluoroquinolones, sparfloxacin provides improved efficacy against important ocular pathogens. Sparfloxacin is effective against Staphylococcus aureus, Streptococcus pneumoniae, and Staphylococcus epidermis. Heamophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumonia, N. gonorrhoea, E. coli, Enterobacter species, Citrobacter species, Shigella species, Salmonella species, Aeromonas species, Yersinia species, Proteus species, Providencia species, M.Catarrhalis, Campylobacter species, S. pyogenes, Haemolytic streptococci, S. pneumoniae and C. trachomatis.
Sparfloxacin inhibits bacterial DNA-gyrase (Topoisomerase II), leading to the disruption of DNA structure and death of the bacteria. This specific mechanism of action eliminates the possibility of plasmid-mediated resistance.
Any ocular pharmacokinetic study has not been reported for sparfloxacin ophthalmic preparation to the best of our knowledge. In order to widen the therapeutic application of sparfloxacin in eye infections, we evaluated the pharmacokinetic of Sparfloxacin 0.3% eye drops in aqueous humour of the rabbit eye.
Materials and Methods
Ocular pharmacokinetic Study
New Zealand white rabbits from the inbred colony of about 5-6 months age and weighing about 1.6-2.2 kg were selected. The eyes of the rabbits were examined to ensure that they did not have abnormality in any ocular structures. The animals were maintained at 22 ± 3 °C temperature and at a humidity of 40 to 60%. All the animal experiments were done in accordance with the OECD principles of good laboratory practice. The approval from Institutional Animal Ethics Committee was taken before initiating the experiments.
The 27 New Zealand white rabbits were selected for the study. They were divided in the 3 groups of 9 rabbits each. First group of nine rabbits were kept in rabbit holder and marked serially. One rabbit was administered 50 µl of saline in the right eye conjuctival sac and other eight rabbits were instilled with 50 µl of sparfloxacin 0.3% ophthalmic preparation in the conjuctival sac. The conjuctival sac was held for 30 seconds with the help of fingers. The aqueous humour (100 µl) was collected from the anterior chamber of the eye with the help of 30 G needle immediately after saline instillation from rabbit 1 and after 15 min., 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr and 6 hr following sparfloxacin 0.3% ophthalmic preparation instillation from other eight rabbits. Just before collection of aqueous humour, rabbits were anaesthesised with intra-muscular injection of xylazine 30 mg/kg and ketamine 5 mg/kg. The other two sets of experiments were carried out in a similar manner.
Analysis of Sparfloxacin in Aqueous Humour
The sparfloxacin concentration of all aqueous humour samples was measured by a modified High Performance Thin Layer Chromatography (HPTLC) technique as described by Mody et al.17 Briefly, the method is described as follows:
The aqueous humour sample (100 µl) was collected in a small glass tube and freeze dried by lyophilisation, (B.Braun Biotech International, Germany). After complete lyophilisation, all residues were redissolved in 100µl dichloromethane by vigorous vortex mixing and 20 µl aliquots of the samples were spotted onto Thin Layer Chromatography (TLC) plates with the help of a Camag Linomat IV autosampler. Sparfloxacin (50ng, 100ng) reference standards were separately spotted on each TLC plates, as the external standard. The TLC plates were developed (10 cm) in a Camag Twin Through glass chamber with a solvent system consisting of chloroform: methanol: formic acid: water (35: 4: 2: 0.25, v/v/v/v), in which the drug had a Rf value of 0.20 + 0.05. Determination of sparfloxacin was done after drying the plates completely using a hot air drier by scanning the fluorescent spots of sparfloxacin at 365 nm on TLC plates with the help of a Camag TLC Scanner 3. Sparfloxacin concentration in samples was calculated with the use of standard curves that were prepared by spiking rabbit aqueous humour with a known amount of sparfloxacin. The detector response was linear over the concentration range of 10-200ng. The recovery of sparfloxacin from aqueous humour samples was 95.2 + 0.98%.
After instillation of sparfloxacin in the eye, it readily crosses the corneal layers and the mean concentration of sparfloxacin 15 minutes after application was found to be 1.4 µg/ml in the aqueous humour. Maximum mean aqueous humour concentration of 3.7 µg/ml was found at 1.3 hours, after which, the level declined exponentially. At 6 hours the levels reduced to 0.56 µg/ml. The mean aqueous humour concentration of sparfloxacin at various time points after instillation of drug into the rabbit eye is shown in figure1. The pharmacokinetic parameters were calculated with help of Winnonlin pharmacokinetic software (Pharsight Corporation, USA) and are shown in Table 1. The peak level (Cmax) and the time taken to reach peak level (tmax) were observed. The elimination rate constant (Kel) and the terminal elimination half-life were estimated by linear regression of the terminal part of the log concentration time curve. The area under the aqueous humour concentration-time curve (AUCall) was determined by the linear trapezoidal rule and extrapolated to infinity (AUC0 ->α) by dividing the last measurable concentration by the elimination rate constant. The mean AUCall was found to be 12.567 ± 0.123 µg/ml/hr. The elimination rate constant was 0.521 per hour and half-life of the sparfloxacin in the aqueous humour was 1.36 hours.
The therapeutic index was calculated by finding the ratio of Cmax: MIC (minimum inhibitory concentration) and AUC: MIC. When the Cmax: MIC ratio is < 1 the antibiotic is considered as ineffective, if it is> 1 but < 2, the antibiotic is considered as bacteriostatic and if the ratio is > 2 it is considered as bactericidal. The Cmax: MIC ratio for sparfloxacin in aqueous humour was 3.7 for Pseudomonas aeruginosa, 7.4 for Streptococcus species, 14.8 for Klebsiella pneumoniae, 29.6 for Staphylococcus aureus, 46.25 for Nisseria gonnorrhoeae and 61.66 for Haemophilus influenzae. The AUC: MIC ratio for sparfloxacin in aqueous was 13.67 for Pseudomonas aeruginosa, 27.35 for Streptococcus species, 54.7 for Klebsiella pneumonia, 109.39 for Staphylococcus aureus, 170.92 for N.gonorrhea and 227.9 for H.influenzae (Table 2).
Fluoroquinolones are widely used in the treatment of bacterial conjunctivitis, bacterial keratitis and other ocular infections. The fluoroquinolones such as ciprofloxacin, ofloxacin, lomefloxacin are shown to have good ocular penetration in humans as well as in experimental animals.18192021
In an ocular pharmacokinetic study in rabbits19 ciprofloxacin (0.3% w/v) and lomefloxacin (0.3% w/v), had a Cmax of 0.103 µg/ml and 1.62 µg/ml respectively at the Tmax of 1 hour. In another comparision lomefloxacin has better penetration than norfloxacin in aqueous humour but was eliminated faster than norfloxacin.22 The aqueous humour concentration of ofloxacin (0.3%) and norfloxacin (0.3%) after topical administration21 is reported to be 1.34 µg/ml and 0.057 µg/ml respectively. With multiple application of ciprofloxacin every 1 hr for 7 and 14 hrs., the mean aqueous humour levels were reported to be 1.31 and 2.18 µg/ml, respectively23 and the mean ofloxacin aqueous humour concentration following similar application was 1.45 and 2.48 µg/ml respectively.24
Combined topical plus oral administration yielded antibiotic concentrations in aqueous of 3.84 ± 2.82 µg/mL ofloxacin compared with 0.35 ± 0.30 µg/mL ciprofloxacin.25
Sparfloxacin is a new fluoroquinolone antibiotic with a broad antibacterial spectrum and good penetration into tissues.26 Previous studies have shown that sparfloxacin has better ocular penetration in aqueous and vitreous humour when given systemically in rabbits as well as in patients undergoing cataract extraction.272829
In the present study the Cmax after single topical application of sparfloxacin was found to be 3.7 µg/ml at the Tmax of 1.33 hour, which are higher than the reported aqueous humour levels of other fluoroquinolones, 2.3 times higher than lomefloxacin, 30 times higher than ciprofloxacin, 2.8 times higher than ofloxacin and several times higher than norfloxacin. This proves superior ocular penetration of sparfloxacin in aqueous humour than other fluoroquinolones.
The aim of antibacterial therapy is to achieve sufficient drug concentrations at the site of infection for an adequate length of time to ensure bacterial eradication and optimise clinical success. The best prediction of clinical outcome depends on the pattern of microbial killing and the persistence of antibacterial effects after plasma or tissue concentrations have fallen below the MIC for target pathogen.30 The beta-lactams generally exhibit time-dependent bacterial killing with minimal persistent effects. Azithromycin, telethromycin, streptogramins and fluoroquinolones exhibit concentration-dependent killing and have prolonged persistent effect. For these agents the AUC: MIC or Cmax: MIC ratio correlates most closely with clinical efficacy.31 The crude MIC values may not represent ideal surrogates for the assessment of clinical antimicrobial efficacy.32 The bactericidal activity is maximized when the ratio of Cmax: MIC or AUC: MIC exceed specific threshold values33. It has been reported that 99% killing can be obtained by quinolones at a low Cmax/MIC ratio, i.e. Cmax/MIC ratio is 3 for ciprofloxacin, and bacterial regrowth and development of bacterial resistance may occur unless higher ratio i.e. Cmax/MIC ratio is reached beyond 8.32 In a recent study, the pharmacokinetics of a ciprofloxacin and sparfloxacin were simulated in vitro and the effects of Pharmacodynamic parameters on bactericidal activity and the emergence of quinolone resistance were examined for Streptococcus pneumoniae. The emergenece of resistance following exposure to ciprofloxacin developed when the Cmax:MIC ratio was less than 4 or the AUC:MIC ratio was less than 10. In contrast the organisms remained clinically susceptible when exposed to higher concentration of sparfloxacin.34 Increase in MICs were noted following exposure of anaerobic bacteria to levofloxacin, trevafloxacin, and sparfloxacin at AUC:MIC ratios of 6 to 14.35
In the present study, the pharmacokinetic parameters were evaluated after measuring the sparfloxacin concentration in aqueous humour. After 15 minutes of instillation of sparfloxacin (0.3%), the aqueous humour level was found to be 1.47 µg/ml, which reaches to the peak level of 3.7 µg/ml at 1.3 hrs and decline to 0.57 µg/ml at 6 hrs. The levels of sparfloxacin in aqueous humour in the present study was sufficiently higher than the MIC reported for common ocular pathogens such as Staph aureus, Staphylococcus epidermis, Streptococcus pneumoniae, H.influenzae, Pseudomonas aeuroginosa, Klebsiella pneumonia and Nisseria gonorrhea.36
The Cmax:MIC and AUC:MIC ratios were calculated for the better prediction for sparfloxacin against common ocular pathogens. The Cmax:MIC ratio was found to be in a range from 3.7 for Pseudomonas to 61.6 for H. influenzae, as evident from table 2. Based on this data we can predict that sparfloxacin can produce 99% bacterial killing against common ocular pathogens and chances for developing resistance is very minimal. The AUC:MIC ratio is also found to be higher for all ocular pathogens. The AUC:MIC ratio was found to be in a range from 13.67 for pseudomonas to 227.9 for H. influenzae.
In conclusion, it can be stated that sparfloxacin has better penetration into the aqueous humour than other quinolones such as ofloxacin, ciprofloxacin and lomefloxacin. Based on the Cmax:MIC and AUC:MIC ratios, sparfloxacin could be clinically effective against Staph aureus, Staphalococcus epidermidis, Strept pneumonia, H. influenzae, Klebsiella pneumonia and N. gonorrhea.
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