Formulation and Stability Studies of Levofloxacin 0.5% Eye Drops
Faris .T. Abachi 1 , Fouad K. Mohammad2and Amar Baba1
1- Department of Pharmaceutical Sciences , College of Pharmacy .
2- Department of Physiology , Biochemistry and pharmacology , College of Veterinary Medicine , University of Mosul , Iraq.
Abstract
The fluoroquinolone levofloxacin is highly effective against gr (+) and gr (-) ocular pathogenic bacteria causing conjunctivitis or corneal ulcers. The ophthalmic preparations of levofloxacin are not produced in lraq as well as active agent.. Therefore , the present report introduce the know-how for the production of levofloxacin 0.05% sterile
ophthalmic solution.
There are multiple dose antibacterial preparations for topical
ophthalmic use for adult and child .The active ingredient is levofloxacin hemihydrate at concentration 0.5 % ( 5.12 mg / ml ) was prepared under aseptic conditions. The fait yellow ophthalmic solution , clear , sterile , isotonic with 308 mOsm / Kg , the pH measured at 6.8. This solution did not cause any eye irritation in rabbits after daily
application for four consecutive days . Finally , the stability of the prepared levofloxacin 0.5 % eye drops was studied at 4 , 25 , 40 and 60 C° for 90 days and calculated the expiration date of the
preparation on was approximately two years . Introduction
Levofloxacin is a third generation fluoroquinolone antibacterial agents with a wide spectrum of activity against both gram positive and
negative microorganisms (1) .It is the active ( S ) isomer isolated from the racemic ofloxacin (2) .
N O N N
F
O
OH O
H3C CH3
1/2 H2O
Levofloxacin Hemihydrate C18H20FN3O4.1/2 H2O
The drug is inhibit of bacterial DNA gyrase ( topoisomerase II ) and topoisomerase IV .These two enzymes are essential for the uncoiling of DNA( 3).
The ophthalmic levofloxacin is indicated in the treatment of corneal ulcers caused by susceptible strains of bacteria . lt is also used in the treatment of conjunctivis caused by susceptible strains of
staphylococcus aureus . Not all species or strains of a particular organism may be susceptible to levofloxacin , streptococcal species are often less susceptible ( 4) .
Recently , we developed levofloxacin ophthalmic solution . However , the stability of this preparation is unknown. Therefore , the present study was undertaken to examine the stability of levofloxacin 0.5 % preparation and to find out the period through which it can be used.
Materials and Methods
Levofloxaci hemihydrate powder from Cord Lab. ( Led ) , India . All solvents were of analar grade and used as received . The prepared o.5% ophthalmic solution of levofloxacin was examined for pH 6.8 ( Philips , UK ) , osomolality using an automatic freezing point
depression osmometer ( Osmomat T 030 , Germany ) , the
concentration of the active ingredient ( levofloxacin hemihydrate ) using high performance liquid chromatography with uv detectot ( Shimadzu , Japan ) , and the wavelenghth λ max measurement using Cary UV-VIS ,100C.
Levofloxacin ( 0.5 % as a base ) ophthalmic solution was prepare under aseptic conditions as fellows in table 1 :
Table 1 : Amount of ingredients found in the prepared ophthalmic solution
Ingredients Amount per 100 ml
Levofloxacin hemihydrate 512 mg
Sodium Chloride 157 mMole/ liter
Benzalkonium chloride 0.05%
0.01N HCI for pH As needed
0.01N NaOH for pH As Needed
Water for injection Add to 100 ml
Dissolve 512 mg of levofloxacin hemihydrate in 90ml in normal
solution 0.9% of sodium chloride add 0.05% of benzalkonium chloride as preservative , measure the pH at 6.8 ,then complete to 100ml of solution . Mix all ingredients ,filter using 0.2μm filter membrane , then measure the osmolality . Pack the solution in small dropper container , sterilized.
The content of the solution can be examined by HPLC techniques.
Mobile phase mixture from water : Butanol : Methanol : Ammonia in ratio ranges 5:5:5:0.4 v/v % respectively.
Standard solution prepare by dissolve a 0.01 mg / ml of levofloxacin hemihydrate in pure water for injection .
Assay preparation : Transfer an accurately measured volume of ophthalmic solution , equivalent to about 6 mg of levofloxacin
hemihydrate , to a 50 ml volumetric flask , dilute with water , and mix . The liquid chromatography is equipped with a 340 nm deter and a 4.6mm * 25 mm in C8 column. The flow rate at about 1 ml / minute . (5)
Separately inject equal volumes of 20 μL of the standard preparation and the assay preparation into the chromatograph , record the
chromatographs and measure the response for levofloxacin
hemihydrate ( C18H20FN3O4.1/2 H2O ) in each ml of the ophthalmic solution taken by the formula : (352.38 / 370.38 ) ( 50C/V )( rμ / rs ) in which 352.38 and 370.38 are the molecular weight of levofloxacin and levofloxacin hemihydrate , C is the concentration in mg / ml of
levofloxacin as standard . V is the volume in ml of ophthalmic solution taken and rμ and rs are the peak responses obtained from the assay preparation , respectively .
Stability Study of the eye drops were incubated at 4 , 25 , 40 and 60 C
° .for 3 months , and the samples were removed later at 30 , 60 and 90 days following incubation.
All experiments were carried out in duplicate and the mean of the results was used.
The rate constants of decay were determined by plotting the log levofloxacin concentration against time , and the energy of activation ( E ) was estimated from the Arrhenius equation ( 6) .
Log K = log A – E / 2.303 RT
Where K is the rate constant of the reaction , A the frequency factor , R the gas constant (1.9872) and the absolute temperature . The method of shelf- life prediction based on three months accelerated stability data was utilized. A graphic technique was employed to predict the degradation that may occure over prolonged storage at 4 , 25 , 40, &
60 C°.
Results
The design of the formula , stability studies for the levofloxacin 0.5%
eye drop should be based on knowledge of the behavior and the properties shown in table 2 :
Table 2 : Summary of physical and chemical properties of levofloxacin 0.5 % eye drop
Test Results
Color Fait yellow liquid
Clearance Free from particles
TLC ( W:B:M:A)(5:5:5:0.4) V/V % Rf = 0.7
pH 6.8
Osmolality 308 mOsmol
Content( HPLC ) 102%
Pharmacological test on Mice &
Rabbits No any redness on both eye
applied three times daily for four consecutive
Sterlity (-) ve , the solution is sterile
The accelerated stability studies should be conducted on the eye drop packed in a plastic closed container , the time and the concentration of the active ingredients studies at four different temperatures for three months. The results shown in table 3 .
Table 3 : Time and concentrations for levofloxacin 0.5 % eye drops at different temperatures.
Time
( Days ) Conc.at
4 C° Conc . at
25 C° Conc. at
40 C° Conc. at 60 C°
30 498.3 498.3 496 485
60 498 496.7 491.6 480
90 495 493.4 486.7 475
The rate constants of decay was determined for 0.5 % levofloxacin eye drop using Arrhenius equation . The rate constant of the reaction will be calculated as shown in table 4 .
Table 4 : Degradation rate constants ( K ) of levofloxacin 0.5% eye drops at four different temperatures.
Temperature ( Kelvin °) K ( month -1 )
277 6.3* 10 -5
298 1.9 * 10 -4
313 6.45 * 10 -4
333 2.1 * 10 -3
Discussion
Levofloxacin , an optically active isomer of ofloxacin is a third generation for flroquinolone agents , a fluorinated quinolone with abroad spectrum of antibacterial activity .The active ( L ) isomer of racemate ofloxacin , has all of excellent features of its parent compound 6 . Some common structure features can be observed among the levofloxacin compound , the methyl group at position 4- methyl in piperazin ring . The chemical structure is differ and mode of action from β -lactam antibiotics and aminoglycosides , and therefore may be active against bacteria resistant to beta- lactam antibiotics (8) .
The eye drop preparation of levofloxacin 0.5 % is the first lraqi
preparation of this kind and the stability studies should be done on this new formulatd ye drops , and known to be very active agents against different types of microorganisms. The antibacterial activity of
levofloxacin has in vitro activity against a wide range of Gram-negative and Gram –positive microorganisms ( 1-4) , and is often bactericidal at concentrations equal to or slightly greater than inhibitory
concentrations but their clinical significance in ophthalmic infections is unknown( 3 &9) . The safety and effectiveness of levofloxacin in
treating ophthalmological infections due to these microorganisms have not been established in adequate and well – controlled trials (10) .
At this time ,only 0.5 % eye drop formulation of levofloxacin hemihydrate solution have been prepared and tested for their
physical , chemical and pharmacological activity as shown in table 1 .The solution is clear liquid , each ml contains 5.12 mg of levofloxacin hemihydrate which equivalent to 5 mg / ml ( 0.5 % ) .The best
preservative used is benzalkonium chloride with other inactives.
The solution should be filtered under aseptic condition 0.2 μm filter ,and the pH adjust at 6.8 , and the solution should be isotonic for ophthalmic purposes.
The testing for stability of drugs contain all appropriate , physical , chemical and biological attribute validated stability including analytical procedures should be applied.,
Stability studies were conducted on levofloxacin 0.5% eye drops incubated at 4 , 25 , 40 and 60 C°, and 90 days following incubation.
The accelerated studies at different temperatures were employed to predict the breakdown that may occurs over prolonged periods of storage at normal shelf conditions.
Levofloxacin concentration in the 0.5 % solution was measured by the high performance liquid chromatography techniques. The rate
constants of decay was determined by plotting the log levofloxacin concentrations against time using Arrhenius equation ( 6 ) .
The Arrhenius plot showed the degradation of levofloxacin 0.5%
solution according to first order kinetics .Besides , the physical and chemical stability during storage period of the solution was monitored.
The degradation rate constants (K) were calculated from slopes of the straight lines. These degradation rate constants are presented in table 4 . To determine the shelf life (6) , ( t10 %) Arrhenius plots were
constructed to predict the degradation rate constants at room
temperature ( K 25 ) for levofloxacin eye drops and was found to equal 0.0547 month -1 , and the predicted shelf life was 1.8 year.The
estimated shelf life is two years of the product ensured extended clinical efficacy under normal storage conditions. Howerever clinical trial of the product is lacking , through it was found to be clinically effective in treating conjunctivitis in mice and rabbits. Further no eye eye irritation was detected in rabbits used for this purpose (10).
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