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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 3  |  Issue : 2  |  Page : 9-13

To Evaluate the In Vitro Transcorneal Penetration of Ofloxaxcin, Pefloxacin, Lomefloxacin and Sparfloxacin in The Presence and Absence of Amniotic Membrane Using Goat Cornea


1 Department of Ocular Pharmacology and Pharmacy, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi-110029, India
2 Department of Pharmacology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi-110029, India
3 Department of Medical Ophthalmology, Retina and Uvea, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi-110029, India
4 Department of Pediatric Ophthalmology and Oculoplasty, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi-110029, India

Date of Web Publication11-Dec-2020

Correspondence Address:
Nihar Ranjan Biswas
Director & Vice-Chancellor, Indira Gandhi Institute of Medical Sciences, Sheikhpura, Patna-800014, Bihar; Department of Pharmacology, All India Institute of Medical Sciences, New Delhi-110029; Director & Vice-Chancellor, Indira Gandhi Institute of Medical Sciences, Sheikhpura, Patna-800014, Bihar
India
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  Abstract 


This study was intended to evaluate the in vitro transcorneal penetration of ofloxaxcin, pefloxacin, lomefloxacin and sparfloxacin in the presence and absence of amniotic membrane using goat cornea. This was an experimental study to estimate transcorneal penetration of four 0.3% fluoroquinolone formulations. In the absence of amniotic membrane, the calculated flux of the transfer in case of ofloxaxcin, pefloxacin, lomefloxacin and sparfloxacin 0.3% were found to be 2.41μg/cm2/hr, 4.45 μg/cm2/hr, 7.4 μg/cm2/hr and 0.968 μg/cm2/hr respectively.
In the presence of amniotic membrane these values changed to 3.38 μg/cm2/hr, 2.15μg/cm2/hr, 5.75 μg/cm2/hr and 2.4 μg/cm2/hr respectively.
The transcorneal permeation studies using goat cornea showed the cumulative release in the following order as lomefloxacin>pefloxacin>ofloxacin>sparfloxacin

Keywords: transcorneal penetration, ofloxaxcin, pefloxacin, lomefloxacin, sparfloxacin


How to cite this article:
Ravi AK, Biswas NR, Velepandian T, Sampangi R, Garg SP, Ghose S. To Evaluate the In Vitro Transcorneal Penetration of Ofloxaxcin, Pefloxacin, Lomefloxacin and Sparfloxacin in The Presence and Absence of Amniotic Membrane Using Goat Cornea. J Indira Gandhi Inst Med Sci 2017;3:9-13

How to cite this URL:
Ravi AK, Biswas NR, Velepandian T, Sampangi R, Garg SP, Ghose S. To Evaluate the In Vitro Transcorneal Penetration of Ofloxaxcin, Pefloxacin, Lomefloxacin and Sparfloxacin in The Presence and Absence of Amniotic Membrane Using Goat Cornea. J Indira Gandhi Inst Med Sci [serial online] 2017 [cited 2021 Dec 7];3:9-13. Available from: http://www.jigims.co.in/text.asp?2017/3/2/9/303153




  Introduction Top


Bacterial endophthalmitis is rare but generally serious desease. It occurs after intraocular surgery, penetrating ocular trauma, and corneal ulcer. Various ocular tissues consist of thin membranes, which are rapidly damaged by injections microorganisms that lead to permanent loss of vision.

Fluoroquinolones became a first choice of drug for prophylaxis and the treatment of anterior segment disease and in endophthalmitis due to its broad-spectrum activity against most of the ocular microorganisms. Ofloxacin, a second-generation fluoroquinolone antimicrobial, has a broad-spectrum activity against Gram-positive organisms such as Staphylococcus aureus, S. epidermidis and Streptococcus pyogenes etc and Gram-negative organisms including Pseudomonas aeruginosa, Enterobacteriacae and Haemophilus influenzae etc. The structural formula of ofloxacin is 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1- piperazinyl)-7-oxo-7H-pyrido [1, 2, 3-de]-1,4-benzoxazime- 6-carboxylic acid. The mechanism of action of ofloxacin is based on the inhibition of DNA gyrase enzyme of the bacteria, which is responsible for DNA replication within the bacterial cell. DNA gyrase belongs to a group of enzymes known as DNA topiosomerase. The chromosome of E. coli consists of about 65 domains, each with an average length of 20μm. All domains are attached to a RNA core. DNA gyrase introduces about 333 negative supercoilings into each domain. This is where fluoroquinolones act, preventing DNA from introducing this negative super helical twist into its strands and thus increasing its special requirement within the bacterial cell. The cell can longer replicate. Ofloxacin possesses an additional killing mechanism along with ciprofloxacin that mostly increases their potency, and may also explain the lower frequencies of mutant resistant to ofloxacin compared with other fluoroquinolones. Ofloxacin is available as a topical 0.3% solution, intravenous preparation and as an oral tablets.

Sparfloxacin, a recently introduced fluoroquinolones antimicrobial for topical use in ophthalmic practices, is more active against staphylococci and appears to penetrate into the non-inflamed vitreous better than ciprofloxacin[1] In experimental study, sparfloxacin showed excellent penetration into the vitreous humour, with mean concentrations in the vitreous of uninflamed eyes of 59.4%±12.2% of that in the serum. Pefloxacin is shown to be widely distributed in the tissues and fluids. After systemic administration in human subject, Pefloxacin has been found in the aqueous humour and lenses of patients undergoing cataract extraction[2].


  Materials & Methods Top


Materials Chemicals Ofloxacin, pefloxacin mesylate dihydrate, and sparfloxacin pure powder were gifted by Cipla Ltd, Mumbai (India) and lomefloxacin hydrochloride powder was gifted by plant Organics Ltd., Hyderabad (India). HPCL grade acetonitrile (E. Merck [India] Ltd, Mumbai), methanol (Ranbaxy fine chemicals Ltd, New Delhi, India), orthophosphoric acid (Spectrochem private Ltd, Mumbai, India) and potassium di-hydrogen orthophosphate (Central drug house (P) Ltd, Mumbai, India) were Procured from commercial sources.


  Eye Drops Top


Oflox 0.3% (ofloxacin) and Proflox 0.3% (pefloxacin) eye drops were gifted by Cipla Ltd, Mumbai (India). Lomibact 0.3% (lomefloxacin) and scat 0.3% (sparfloxacin) were gifted by Milmet Pharma Ltd., Baroda (India) and Sante vision, Division of Cadila Pharma Ltd., Ahmedabad (India) respectively.


  Amniotic Membrane Top


Processed and frozen human amniotic membrane preserved in Dulbecco’s modified eagle medium (DHEM) and dimethyl sulfoxide (1.1) was procured from National Eye Bank, Dr. R P Centre for Ophthalmic Sciences, AIIMS, India and stored in deep freezer at -70º_C temperature. Before using, it was thawed at ambient temperature. Saline wash was given just prior to grafting over the cornea.


  Goat Eyeball Top


Goat eyes were procured from the local slaughter house. Transcorneal Penetration Assembly

Four cell diffusion chamber was fabricated in our laboratory to conduct in vitro transcorneal penetration study.


  HPCL System Top


High performance liquid chromatography (HPCL) was used for quantification of the drugs in aqueous humour samples. Thermo Finnigan HPCL system (Thermo Electron Crop, USA) consisted with a quaternary gradient surveyor pump (Thermo Electron Corp, USA), photodiode array (PDA) detector, auto-sampler coupled with Rhyodyne injector, online degasser was used. The spectral match was achieved using inbuilt PDA library option using ChromQuest (Ver 4.1) software (Thermo Electron Corporation, USA).


  Methods Top


Preparation of simulated physiological buffer solution

106.5 mM - Sodium chloride

26.1mM - Sodium hydrogen carbonate

0.5 mM - sodium dihydrogen phosphate

1.1mM -Calcium chloride

10 mM - Hepes buffer 18.7 mM - Potassium chloride

1.0 mM - Magnesium chloride

The above said ingredients were dissolved in freshly prepared double distilled water and the resulting fluid was showing the pH 7.4.

In vitro Transcorneal Permeation Studies

In vitro transcorneal permeation was carried out by using modified Franz’s diffusion chamber. The whole system consisted of 4 cells and each cell consisted of upper and lower chambers. The goat cornea was placed in between these two chambers. The upper chamber acted as a donor compartment in which 50μl of 0.3% ofloxacin, perfloxacin, l o m efloxacin, sparfloxacin and nanoparticulated sparfoxacin formulation was placed. The lower chamber acted as a receptor compartment, which was pefused continuously with physiological buffer solution (shown above) at the rate of 1 ml/hr. The whole system was maintained at 36º_c.

In another set of experiment, human amniotic membrane was spread above the goat cornea and this preparation was placed in the diffusion chamber to study the permeation of individual fluoroquinolones in presence of amniotic membrane.

Sample Collection

Perfusate was collected in a pre-weighed tubes at every 15 mins interval for the total period of 2 hrs. After collection, the tubes were reweighed and the volume was determined via density calculation. The Collected samples were stored at -70º¬C till further analysis by HPLC.


  Sample Analysis Top


Assay Condition

The analytical separation of individual fluoroquinolones was achieved with different proportions of the mobile phase, which consisted of 0.02M potassium dihydrogen phosphate adjusted to pH 2.5 and acetonitrile (CAN) [Table A]. 0.22μ filtered solvents and buffer were degassed with in-built on-line degasser and pumped at the flow rate of 1 ml/min. The column temperature was kept at 25±1º_C. The ambient temperature was maintained at in and out of the system. Twenty microliter of sample was injected from 96 well sample plates using auto-sampler and the analytical separation was achieved using RP-C18 and C8 column of different make at particular wavelengths for individual fluoroquinolones as shown in the [Table A].
Table A: Mobile phase Ratio, Column and Wavelength for Individual FQs

Click here to view


Standard Solutions

Stock solutions of ofloxacin (1 mg/ml), pefloxacin (1mg/ml), sparfloxain (1 mg/ml) and lomefloxacin (1mg/ml) were prepared by dissolving each drug in pure methanol (HPLC grade). Working standard solutions of the drugs were made with 50% methanol-water.

Extraction Procedure

All the perfusate samples were processed/ deproteinated with acetonitrile (ACN) in the ratio of 1:1 (sample: ACN) before the quantification of the drug by HPLC.


  Calculations and Statistics Top


Calculation

For in vitro penetration studies on fluoroquinolones in the presence or absence of amniotic membrane, the flux of the individual drug was calculated from the slope of the linear portion of the curve plotted against cumulative amount of drug vs time.

The apparent permeability coefficient (Papp, cml/sec) for the fluoroquinolones under study was calculated by using the following formula.

Papp =F/S * CO

Where,

F = Flux calculated from the slope of the linear portion of the permeability curve

S = Surface area exposed for the drug permeation (0.6 cm2)

Co = Initial concentration of drug loaded in the donor compartment at t=o


  Statistical Analysis Top


For the evaluation of significance, appropriate statistical methods were adapted by using the software sigma Stat (Ver.2) and p value <0.05 was considered for the level of significance. The results are expressed as Mean±SD (Standard Deviation).


  Results Top


Transcorneal penetration of ofloxacin 0.3%

After the application of 150 μg of ofloxacin in the volume of 50μl on the donor compartment, showed a cumulative transfer of 4.2 μg/ cm2 over the period of 2 hrs in the acceptor compartment. This amounted to be 2.8% of the applied concentration of the ofloxacin over the cornea. The calculated flux for this transfer was found to be 2.41 μg/cm2/hr.

In the presence of amniotic membrane this transfer rate was enhanced and was increased to 5.78μg/cm2. Amniotic membrane enhanced the transfer of the ofloxacin to 1.4 fold. The calculated flux for this transfer was found to be 3.38 μg/cm2/hr.

Transcorneal Penetration of Pefloxacin 0.3%

Application of 150 μg of pefloxacin in the volume of 50 μl on the donor compartment showed a cumulative transfer of 7.5 μg/cm2 over the period of 2 hrs in the acceptor compartment. This amounted to be 5% of the applied concentration of the pefloxacin over the cornea. The calculated flux for this transfer was found to be 4.45 μg/cm2/hr.

In the presence of amniotic membrane, this transfer rate was affected and was reduced to 4.2 μg/cm2. Amniotic membrane offered 1.78 fold resistance for the transfer of the pefloxacin. The calculated flux for this transfer was found to be 2.15 μg/cm2/hr.

Transcorneal penetration of Lomefloxacin 0.3%

When 150 μg of lomefloxacin was applied in the volume of 50 μl on the donor compartment, acceptor compartment showed a cumulative transfer of 13 μg/ cm2 over the period of 2 hrs. This amounted to be 8.6% of the applied concentration of lomefloxacin over the cornea. The calculated flux for this transfer was found to be 7.4μg/cm2/hr.

In the presence of amniotic membrane, this transfer rate was affected and was reduced to 11.5 μg/cm2. Amniotic membrane offered comparatively less resistance of 1.1 for the transfer of the lomefloxacin as compared to the compared to the control. The calculated flux for this transfer was found to be 5.75 μg/cm2/hr.

Transcorneal penetration of Sparfloxacin 0.3%

After the application of 150 μg of sparfloxacin in the volume of 50 μl on the donor compartment showed a cumulative transfer of 1.7 μg/cm2 over the period of 2 hrs in the acceptor compartment. This amounted to be 1.2% of the applied concentration of the sparfloxacin over the cornea. The calculated flux for this transfer was found to be 0.968 μg/cm2/hr.

In the presence of amniotic membrane, this transfer rate was facilitated and was increased to 3.8 μg/cm2. Amniotic membrane enhanced the transfer of the sparfloxacin to 2.2 fold. The calculated flux for this transfer was found to be 2.4 μg/cm2/hr.


  Discussion Top


Fluoroquinolones are a class of synthetic antibacterial agents that were approved for ocular therapy in 1991. Ever since they have been used as a popular therapy for the treatment and prevention of various ocular infections.[3] Presently, fluoroquinolones have been widely used as an antibacterial agent with topical, intravitreal and systemic routes of administration. These agents are synthetic,with broad-spectrum of activity, rapidly bactericidal, and good penetration into ocular tissues.[4] In general, fluoroquinolones such as norfloxacin, ciprofloxacin, ofloxacin, and lomefloxacin etc. have good ativity against Gram-positive bacteria. Therapy with fluoroquinolones has largely replaced the combination therapy for main ocular infections e.g. bacterial keratitis as the therapeutic concentrations are well achieved in the cornea after topical administration. These agents are not reported to cause corneal toxicity or retinal toxicity when used in therapeutic topical or systemic dosages.

Corneal precipitation occurs, particularly with ciprofloxacin and to a lesser extent norfloxacin, but does not appear to interfere with healing of epithelium. In treatment for endophthalmitis, there is a reasonable penetration of systemic fluoroquinolones into the vitreous humour but sufficiently high concentrations to reach the minimum inhibitory concentration for 90% of isolates (MIC90) of all important micro-organisms may not be guaranteed. Systemic administration is useful for prophylaxis after ocular trauma. Still, it is not sure that how much of systemically given fluoroquinolones would be reaching in intraocular fluids for the treatment of endophthalmitis. Several studies reported the individual ocular pharmacokinetics of topically, subconjunctivally, systemically administered fluoroquinolones. However, due to the difference in the clinical set up and protocol used for these studies, it is very difficult to compare their ocular pharmacokinetics and pharmacodynamics together.

Therefore, a constant desire has been felt to evaluate and compare the commonly used fluoroquinolones for their ocular penetration after oral, topical and intravenous modes of administration.

Thus in this purview, the present study was planned to compare the pharmacokinetics profile with pharmacodynamics effects of commonly used ocular fluoroquinolones. This would definitely help to compare and evaluate the safety and efficacy of these agents by in vitro, in vivo and clinical settngs.

The first use of amniotic membrane transplantation in ophthalmology was done by the De Rotth in 1940, who reported partial success in the treatment of conjunctival epithelial defects after symblepharon (scarring and adhesions between palperbral and bulbar conjunctiva). Amniotic membrane may be used as “filler” for closing corneal perforations. Ever since, several articles appeared stating the usefulness of amniotic membrane grafting (AMG) in various ocular conditions.5 Increasingly, AMG is employed in various conditions like ulceration in the cornea and sclera, the treatment of difficult disorders such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid, recurrent pterygia, Persistent Epithelial Defects (PEDs), acute chemical and thermal injuries, shield ulcer of vernal keratoconjunctivitis,[6] neurotrophic keeratitis, and partial stem cell deficiency. However, the constraints exerted by AMG, which in turn affects the transcorneal penetration of topically applied fluoroquinolones were not studied in detail. In many conditions, ophthalmologists are having doubts about the trans-amniotic penetration of topically administered antimicrobial agents. Therefore, the present study was also planned to study the in vitro penetration characteristics for the topically applied fluoroquinolones in presence and absence of amniotic membrane.

To study the in vitro penetration characteristics of fluoroquinolones, corneal permeation chamber was specially fabricated with a continuous receptor compartment replacement system. The chamber was maintained at normal corneal temperature of 36º_C. The model was a modified flow cell having a continuous infusion of lower compartment, in which a replaceable volume of 0.47cm3 ,through which physiological buffer solution was perfused. The system was an alternative model to Franz’s diffusion cell, which maintained a static acceptor compartment. Goat cornea was selected to study the permeation characteristics because of it’s easy availability in the local slaughter house and also as it decreases the requirement of sacrificing a rabbit only for cornea. Increasing use of amniotic membrane in ophthalmologic practice raised many questions regarding its permeability characteristics for the corneal transfer of drugs.

In the present study, four goat corneas were used for each experiment. We compared four fluoroquinolones (0.3% ofloxacin, 0.3% lomefloxacin and 0.3% sparfloxacin in the volume of 50μl each) for penetration through cornea individually. In the other group, cornea was completely covered by amniotic membrane (AM) and individually 0.3% ofloxacin, 0.3% lomefloxacin and 0.3% sparfloxacin were studied.

In this in vitro study, we observed that amniotic membrane facilitated the corneal penetration for all studied drugs when compared to control. Among four studied drugs, the mean cumulative release was found to be maximum for lomefloxacin followed by pefloxacin, ofloxacin and sparfloxacin respectively. Amniotic membrane increased the mean Cumulative release in sparfloxacin and nanoparticulated formulation of sparfloxacin and decreased the cumulative release in pefloxacin group. Marginal increase and decrease in mean cumulative release were observed in ofloxacin and lomefloxacin respectively.

A similar study was conducted by Fukuda et al.[7] reported that the apparent permeation coefficient of ofloxacin as 6.77×10-7 cm/s in rabbit cornea. In our, in vitro ofloxacin was found to be 4.46×10-6cm/s. The difference in values could be due to the variation in the species.

However, contrary to this Tai et al.[8] determined the corneal and sclera permeability coefficient of ciprofloxacin, cinoxacin, enoxacin and ofloxacin in rabbits using high performance liquid chromatography. They have reported that corneal permeability coefficient in rabbits among ciprofloxacin, norfloxacin, cinoxacin, enoxacin, and ofloxacin were not significantly different. But the results of our study concluded that there is a difference in the permeability coefficient among the fluoroquinolones studied viz ofloxacin, pefloxacin, lomefloxacin and sparfloxacin in presence or absence of AMG.


  Conclusion Top


This study has been planned to comparatively evaluate the transcorneal permeation of 0.3% w/v of fluoroquinolones like ofloxacin, pefloxacin, lomefloxacin and sparfloxacin using goat cornea. Considering the increased use of amniotic membrane in the ophthalmic practice, this study was extended to evaluate their permeation characteristic in the presence of amniotic membrane. Its permeation characteristics were studied on in vitro transcorneal permeation assembly.

These studies resulted the following conclusion: The transcorneal permeation studies using goat cornea showed the cumulative release in the following order as lomefloxacin>pefloxacin>ofloxacin>sparfloxacin. In the presence of amniotic membrane the corneal penetration for all studied drugs were altered as compared to their control. The cumulative release was increased in sparfloxacin group and decreased in the pefloxacin group. Marginal increased and decrease in mean cumulative release were observed with ofloxacin and lomefloxacin respectively. The results of this study further concluded that there is a difference in the permeability coefficient among the fluoroquinolones studied viz ofloxacin, pefloxacin, lomefloxacin and sparfloxacin.



 
  References Top

1.
Cochereau-Massin I, Bauchet J, Marrakchi-Benjaafar S, Saleh-Mghir A, Faurisson F, Vallois JM, Vallee E, Pocidalo JJ. Efficacy and ocular penetration of sparfloxacin in experimental streptococcal endophthalmitis. Antimicrob Agents Chemother 1993;37(4):633-6.  Back to cited text no. 1
    
2.
Salvanet A, Fisch A, Lafaix C, Montay G, Dubayle P, Forestier F, Haroche G. Pefloxacin concentrations in human aqueous humour and lens. J Antimicrob Chemother 1986;18(2):199- 201.  Back to cited text no. 2
    
3.
Andersson MI, MacGowan AP. Development of the quinolones. J Antimicrob Chemother 2003, 51(1): 1-11.  Back to cited text no. 3
    
4.
Stroman DW, Dajcs JJ, Cupp GA, Schlech BA. In vitro and in vivo potency of moxifloxacin and moxifloxacin ophthalmic solution 0.5%, a new topical fluoroquinolone. Surv Ophthalmol 2005 ;50(1): S16-31.  Back to cited text no. 4
    
5.
Bajaj MS et al. Evaluation of Amniotic Membrane Grafting in the Reconstruction of Contracted Socket. Ophthal Plast Reconstr Surg 2006, 22(2), 116-120.  Back to cited text no. 5
    
6.
Sridhar MS et al. Amniotic Membrane Transplantation in the Management of Shield Ulcers of Vernal Keratoconjunctivitis. 2001, 108(7): 1218-22.  Back to cited text no. 6
    
7.
Fukada, M and Sasaki. In vitro topically applied fluoroquinolone penetration into the anterior chamber. Nippon Ganka Gakkai Sasshi 1995, 99:532-36.  Back to cited text no. 7
    
8.
Tai MC, Lu DW, Chiang CH. Corneal and Scleral permeability of quinolones - a pharmacokinetics study. J Ocul Pharmacol Ther. 2003;19:547-54.  Back to cited text no. 8
    



 
 
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  In this article
Abstract
Introduction
Materials & ...
Eye Drops
Amniotic Membrane
Goat Eyeball
HPCL System
Methods
Sample Analysis
Calculations and...
Statistical Analysis
Results
Discussion
Conclusion
References
Article Tables

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