- Email: [email protected]
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride
J. DRUG DEL. SCI. TECH., 15 (3) 249-252 2005
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal* Division of Pharmaceutics, Department of Pharmaceutical Technology, Jadavpur University, Jadavpur, Kolkata, PIN 700 032, India *Correspondence: [email protected] Ocular inserts containing ciprofloxacin hydrochloride (CPF) as a model drug were developed by optimization of design applying ANOVA. The parameters of optimization were taken as the concentration of calcium chloride solution and time of exposure of the treatment. Out of the four formulations studied, types III and IV contained hydroxypropylmethylcellulose (HPMC) whereas types I and II were developed without HPMC. Type I and III contained CPF as the soluble hydrochloride salt while in types II and IV CPF is present in crystal form. In vitro dissolution studies with the four formulations showed that time of 90% drug release for types II and IV were 23.8 ± 0.2 and 25.8 ± 2.6 h, respectively, whereas the same for types I and III were 8.9 ± 0.6 and 16.6 ± 1.5 h. In vivo studies on all the formulations in the rabbit eye revealed that tear concentrations of CPF lasted for more than 2 days with types II and IV inserts. All the formulations showed excellent retention properties in the cul-de-sac of rabbit eyes with almost no irritation. Key words: Ocular insert – Factorial design – Ciprofloxacin – Swelling index.
A significant challenge in drug delivery is the local administration of drugs to the eye [1]. The tendency of the eye to drain out instilled fluid volume in excess of 7 to 10 µl severely compromises the performance of liquid dosage forms such as solutions, suspensions and liposomes. Drugs from these dosage forms typically disappear from the cul-de-sac within 1 to 2 min of instillation in human beings and 5 to 10 min in rabbits [2, 3]. Ciprofloxacin hydrochloride (CPF) 0.3% w/v ophthalmic solution, one to two drops at a time, to the affected eye is administered to patients suffering from bacterial infections. Continuous medication for three consecutive days is required to reduce the bacterial conjunctivitis [4] and bacterial keratitis [5] in 95% of the patients. The in vitro minimum inhibitory concentration (MIC90) of CPF hydrochloride is 2 µg/ml [6] against all bacteria causing ocular infections. However, the treatment schedule described above gives a steady state concentration which is much above the MIC90 that may be uncomfortable to the patient, causing severe irritation due to its cationic nature. CPF also produces a white layer of crystals on the ulcerated portion of the cornea [5]. For these reasons, an ocular insert of CPF hydrochloride providing a concentration sufficiently above MIC90 in the tears, will have higher efficacy, prevent loss of drug and produce better patient compliance compared to eye drops. An attempt has been made in the present work to design an ocular insert that will maintain MIC90 of drug in the tears for as long as three days and also provide minimum swelling of the insert during its residence time in the cul-de-sac of the eye [7]. To achieve these objectives, CPF hydrochloride was incorporated in a hydrophilic matrix of sodium alginate film, cross-linked with calcium chloride to reduce swelling to the minimum and to provide prolonged release of the drug.
2. Methods
2.1. Preparation of ocular inserts Type I inserts were prepared by dissolving 25 mg of CPF hydrochloride and 25 mg of glycerol as plasticizer in 6 ml of distilled water, followed by dissolving 250 g of sodium alginate in the same solution. The homogeneous mass was cast on a stainless steel surface and then dried at 45°C for 8 h under an infrared heat source. The dried alginate films were removed and discs of 4.5 mm diameter were punched out with a single punching instrument. The discs were then treated with 1 to 4% w/v of calcium chloride solution for 15 to 60 s followed by blotting with filter paper to remove excess calcium chloride solution from the surface. The discs were finally air-dried for 1 h. For type II inserts, the solvent used to dissolve the drug and glycerol was a mixture of 5 ml of distilled water and 1 ml of Sorensenʼs phosphate buffer pH 7.4, which yielded a suspension of CPF, which was swirled with 250 mg sodium alginate to prepare a homogeneous, viscous mass. Type III inserts were prepared similar to type I but contained a suspension of 50 mg of HPMC and 200 mg sodium alginate dispersed in it. The type IV inserts were prepared in the same way as type II, but contained 50 mg of HPMC and 200 mg sodium alginate dispersed in it. The remaining steps for types II, III and IV are the same for type I. However, treatment with calcium chloride solution was done for type II, III and IV with 4% w/v solution and exposure time was only 15 s. 2.2. Experimental design In this study, a 22-factorial design [8] was adopted for type I inserts. The effects of two factors – (A) concentration of calcium chloride solution and (B) time of treatment – were studied at two levels: one at higher and the other at lower level since both controls the degree of cross-linking. Four experimental inserts were denoted as (I): 2% w/v CaCl2 concentration and 15 s time of exposure, a: 4% w/v and 15 s, b: 2% w/v and 60 s and ab: 4% w/v and 60 s. The area expansion index (α) of the inserts were measured. Swelling of the inserts in all dimensions should be minimum after prolonged contact to tears. The experiments were carried out in triplicate. The expansion indices were determined by placing one insert in each eye of albino rabbits weighing between 1.5 to 2.0 kg, and taking out after three days to observe their physical characteristics after bloating carefully. The inserts were scanned at 100 dpi (Canoscan N676U) against a black background and the images were pasted on a software made with
I. MATERIALS AND METHODS 1. Materials
Ciprofloxacin hydrochloride was obtained as a gift from Astra IDL, India. Sodium alginate, hydroxypropylmethylcellulose K4M, disodium hydrogen orthophosphate and sodium dihydrogen orthophosphate were purchased from Loba Chemie. Sorensenʼs modified isotonic phosphate buffer pH 7.4 was prepared and used wherever applicable because the rabbit lachrymal secretion has a pH of 7.4. In all experiments, glass double-distilled water was used. All other reagents and solvents were of analytical reagent grade. 249
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal
J. DRUG DEL. SCI. TECH., 15 (3) 249-252 2005
Visual Basic 6.0 programming language. The area of each insert was measured at the beginning and the end of study by checking the hue of each point that is different from the background color.
II. RESULTS AND DISCUSSION 1. Swelling studies of various treatment combinations of inserts
The extent of swelling of type I inserts in rabbit eyes, depicted in Table I, were measured from the projected diameter. It revealed that inserts treated with 4% calcium chloride solution produced less swelling than with 1% calcium chloride solution. The initial diameter of the inserts before treatment was 4.1 ± 0.03 mm. Just after treatment, the diameters of inserts obtained from treatment combination I showed the greatest swelling (4.72 ± 0.18 mm) while treatment combination ab produced minimum swelling (4.18 ± 0.08 mm). Both treatment combinations a and ab produced negligible and almost similar degree of swelling after treatment. Three days after remaining in contact with tears in the cul-de-sac of rabbit eyes, inserts with treatment combinations a and ab exhibited similar swelling indices and were sufficiently lower than treatment combinations I and b. The results of ANOVA resolves the variability of area expansion index into contributions due to two factors, i.e. concentration of calcium chloride and time of treatment. The contribution of each factor is measured having removed the effects of all other factors. Since the P value for the factor concentration of calcium chloride solution is less than 0.05, this factor has a statistically significant effect on area expansion index at a confidence level of 95%. Since treatments I and b produced inserts that were deformed after three days, the concentration of calcium chloride solution was taken at 4% w/v level instead of 1% w/v. Flexibility of the inserts were found to reduce if they were cured for 60 s at 4% w/v concentration of calcium chloride solution. So it was decided that the optimum levels of the factors were 4% w/v calcium chloride solution and exposure for 15 s, i.e. treatment combination a.
Area expansion index = [(final area - initial area)/initial area] x 100 Projected diameter dp = √(4 x area)/π Diameter expansion index = [(final dp - initial dp)/initial dp] x 100 2.3. Determination of drug content One insert was placed in 5 ml of 0.1 N sodium hydroxide solution to dissolve calcium alginate followed by neutralization with addition of 0.1 N hydrochloric acid. After filtration, the solution was assayed by UV spectrophotometer (Beckman DU-64) at 274 nm against a blank prepared similarly with an insert without drug. 2.4. In vitro drug release study The inserts were placed in 10 ml of Sorensenʼs modified isotonic phosphate buffer pH7.4 solution in 20-ml vials with rubber stopper, and then placed in an incubator, maintained at 37.0 ± 0.1°C. Onemillilitre sample was withdrawn and 1 ml of fresh phosphate buffer preheated to 37°C was replaced in the vial at the same time. Absorbances of the samples were measured using a Beckman DU-64 UV spectrophotometer at 274 nm after proper dilution against a blank of fresh phosphate buffer. 2.5. In vivo release study All animal experiments were conducted with prior approval of Institutional Animal Ethics Committee of Jadavpur University, Calcutta. Albino rabbits weighing between 1.5 to 2.0 kg, with no ocular pathology were selected for in vivo studies. In each eye, a sterile insert containing drug equivalent to 150 µg CPF hydrochloride was placed in the cul-de-sac. At appropriate time intervals of 1, 4, 8, 12, 24, 36, 48, 60 and 72 h, tear samples were collected by a capillary tube of internal diameter 383 µm having 1 µl mark lightly placed near the canthus of the eye without applying pressure. In case of conventional eye-drop, one drop of CPF hydrochloride 0.3% w/v ophthalmic solution was instilled in each eye with the help of a drop dispenser. Tear samples were collected at 0.25, 5, 10, 15, 30, 45 and 60 min after instillation. The concentration of CPF was determined by HPLC method [8] using a HPLC system (Perkin Elmer LC200) consisting of a series 200 pump and UV-Vis detector with a NCI 900 network chromatography interface and software (Total Chrome). The mixture of 87 volumes of 0.025 M phosphoric acid, previously adjusted with triethanolamine to a pH 3.0 ± 0.1 and 13 volumes of acetonitrile was used as the mobile phase at a flow rate of 1.5 ml/min and the detector wavelength at 278 nm.
2. In vitro drug release profile with treatment combination a
The release profile of drug from four types of inserts with treatment combination a are shown in Figure 1. Each experiment was performed in triplicate. In vitro parameters of four types of insert are depicted in Table II. Prolonged release of drug from type II and type IV inserts were evident from their time for 90% drug release, i.e. 23.8 ± 0.2 and 25.8 ± 2.6 h, respectively. Both contain crystals of CPF and were entrapped within the calcium alginate matrix. Dissolution of CPF from its crystals was the rate-limiting step because CPF has limited solubility in release medium of pH 7.4 buffer. The times for 90% drug release from type I and type III were much less compared to type II and IV inserts which appears due to the fact that they contain CPF in soluble hydrochloride form. However, in type III, higher entrapment of CPF hydrochloride was obtained due to the presence of HPMC that has possibly formed
Table I - Expansion indices of type I inserts after treatment and after three days in contact with tears. Parameters
(I)
a
Area before treatment (mm2) Projected diameter before treatment (mm) Area after treatment (mm2) Projected diameter after treatment (mm)
b
ab
16.90 ± 1.24
13.75 ± 0.54
13.40 ± 0.20* 4.13 ± 0.03** 17.53 ± 1.30
13.87 ± 0.70
4.72 ± 0.18
4.20 ± 0.11
4.64 ± 0.17
4.18 ± 0.08
Area after 3 days in contact with tear (mm2)
82.80 ± 14.48
37.76 ± 2.10
56.98 ± 14.44
36.12 ± 3.39
Projected diameter after 3 days in contact with tear (mm)
10.24 ± 0.90
6.93 ± 0.19
8.47 ± 1.12
6.78 ± 0.32
Area expansion index after 3 days in contact with tear (%)
372 ± 83
172 ± 15
237 ± 85
163 ± 25
Diameter expansion index after 3 days in contact with tear (%)
117 ± 19
65 ± 5
83 ± 24
62 ± 8
*Mean ± SD, n = 3. **Mean ± SD, n = 6. I, a, b and ab are treatment combinations. 250
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal
J. DRUG DEL. SCI. TECH., 15 (3) 249-252 2005
1400
100
Type-I Type-II Type-III
80
Type-IV
1000 Tear concentration (µg/ml)
% of drug released
1200
60 40 Type-I Type-II Type-III Type-IV
20
800
600
400
0 0
40
20
60
200
Time (h) 0
Figure 1 - Percentage of drug released versus time from type I, type II, type III and type IV inserts treated with 4% calcium chloride solution for 15 s. Mean ± SD, n = 3.
0
10
20
30
40
50
60
70
Time (h)
Figure 2 - Tear concentration (µg/ml) of CPF hydrochloride versus time profile after introducing inserts in the cul-de-sac of rabbit eyes. Mean ± SD, n = 3.
a gel structure in the calcium alginate matrix entrapping the dissolved drug molecules in it. The prolongation of drug release from type III was probably due to the gel structure of HPMC through which the movement of drug molecule was retarded compared to that from type I inserts. The linear regression coefficients of zero-order, Higuchi and firstorder models for drug release were calculated from data up to the time for 90% release. The regression coefficients showed that all types of insert correlate more with Higuchi (mean R = 0.9876) and first-order (mean R = 0.9828) models than with zero-order model (mean R = 0.9610).
completely disappearing after 30 min due to drainage and dilution with tears. Therefore, one drop is required every 15 min to maintain the drug level in tears. The in vivo release profiles of drug from all the four inserts were obtained and presented as tear concentration versus time in Figure 2. All experiments were conducted in triplicate and plotted as mean ± standard deviation. The pharmacokinetic parameters calculated from the in vivo data are represented in Table III. Tear concentration from all three inserts reached maximum value within 2.5 h, except type III which occurred in 11.3 ± 1.2 h. Type I inserts showed the highest peak tear concentration (1242.7 ± 99.4 µg/ml) among all the inserts followed by type IV (1040.3 ± 108.0 µg/ml). The quick release and high peak concentration of CPF in type I inserts were probably due to the fact that CPF was present in the dissolved salt state. The mean residence times (MRT) of drug in all four types of inserts were si-
3. In vivo drug release studies in the rabbit eye
Tear concentrations of CPF hydrochloride were measured for between 15 s and 60 min after instillation of one drop of the ophthalmic solution. The results revealed that the drug disappeared from the cul-de-sac within 30 min after instillation of the drop, the concentration of the drug reducing from 3,000 to 2,100 µg/ml within 15 s and Table II - In vitro parameters of various types of inserts (mean ± SD, n = 3). Parameters
Type I inserts
Type II inserts
Type III inserts
Type IV inserts
Drug content (µg/insert)
137.0 ± 9.2
131.3 ± 8.3
162.5 ± 7.2
129.3 ± 9.8
Time of 90% release (h)
8.9 ± 0.6
23.8 ± 0.2
16.6 ± 1.5
25.8 ± 2.6
0.9481 ± 0.0110
0.9706 ± 0.0057
0.9888 ± 0.0008
0.9366 ± 0.0436
R of zero-order model R of Higuchi model
0.9844 ± 0.0044
0.9968 ± 0.0021
0.9934 ± 0.0060
0.9757 ± 0.0144
R of first-order model
0.9788 ± 0.0168
0.9909 ± 0.0042
0.9831 ± 0.0160
0.9784 ± 0.0120
R of zero-order model = correlation coefficient between percentage of drug released and time. R of Higuchi model = correlation coefficient between percentage of drug released and square root of time. R of first-order model = correlation coefficient between logarithm of percentage of drug remaining to be released and time. Table III - Pharmacokinetic parameters after tear concentration (µg/ml) of CPF hydrochloride after introducing conventional drops and inserts in the cul-de-sac of rabbits. Parameters
Conventional CPF solution (0.3% w/v)
Type I inserts
Type II inserts
Type III inserts
Type IV inserts
Cmax (µg/ml)
-
1242.7 ± 99.4
680.7 ± 4.0
737.0 ± 836
1040.3 ± 108.0
Tmax (h)
-
2.7 ± 1.2
2.5 ± 0.9
11.3 ± 1.2
1.7 ± 0.6
MRT (h)
4.8 ± 0.5 min
8.5 ± 0.3
16.7 ± 0.5
12.9 ± 0.3
14.1 ± 0.8
Duration of action (h)
29.7 ± 1.2 min
38.1 ± 1.6
53.3 ± 0.6
44.0 ± 3.5
56.9 ± 6.9
Cmax = maximum concentration in tears. Tmax = time for the drug to reach Cmax. MRT = mean residence time = area under the first moment curve vs. time/area under the tear conc. curve vs. time. Duration of action = duration in which the concentration of drug remained above MIC90 (i.e. 2 µg/ml). 251
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal
J. DRUG DEL. SCI. TECH., 15 (3) 249-252 2005
gnificantly higher (P < 0.01) than conventional drops as depicted in Table III. Duration of action was assumed as the time for which the tear concentration remained above MIC90, i.e 2 µg/ml. It was found that conventional drops produced duration of action of only 29.7 min whereas durations of action were 38.1 and 44 h with type I and type III, respectively, and 53.3 and 56.9 h with types II and IV. Therefore, type II and type IV exhibited similar duration of action and a single insert is sufficient to produce the necessary therapeutic action for at least two days. Since the peak concentration is minimum (680.7 ± 4.0 µg/ml) with longer duration of action in type II inserts, it is thought to be the best formulation of the four types of insert studied. All of the ocular inserts showed excellent retention properties in the cul-de-sac of the rabbit with minimum swelling of the inserts. They did not come out even after three days. However, with the type II inserts the desirable properties were obtained with duration of action lasting for more than two days and not very high peak concentration of CPF, which might cause discomfort. Tear secretion and blinking rate in the rabbits were found to be normal after introducing the inserts, which indicates minimum or no irritation.
REFERENCES 1. 2. 3. 4. 5.
6. 7. 8.
PRAUSNIT M.R., NOONAN J.S.- Permeability of cornea, sclera and conjunctiva: A literature analysis for drug delivery to the eye.- J Pharm. Sci., 87, 1479-1488, 1998. LEE V.H.L., ROBINSON J.R.- Review: Topical ocular drug delivery: Recent developments and future challenges.- J. Ocular. Pharmacol., 2, 67-108, 1986. LEIBOWITZ H.M.- Antibacterial effectiveness of ciprofloxacin 0.3% ophthalmic solution in the treatment of bacterial conjunctivitis.- Am. J. Ophthalmol., 112, 295-335, 1991. LEIBOWITZ H.M.- Clinical evaluation of ciprofloxacin 0.3% ophthalmic solution for treatment of bacterial keratitis.- Am. J. Ophthalmol., 112, 345-475, 1991. DIAMOND J.P., WHITE L., LEEMING J.P., HOH H.B., EASTY D.L.- Topical 0.3% ciprofloxacin, norfloxacin, and ofloxacin in treatment of bacterial keratitis: a new method for comparative evaluation of ocular drug penetration.- Br. J. Opthalmol., 79, 606-609, 1995. DAROUGAR S., DAROUGAR D.- US Patent 6246971- East Croydon, GB. MONTGOMERY D.C. -The 2k factorial design.- In: Introduction to Statistical Quality Control, D.C. Montgomery Ed., John Wiley & Sons Inc., New York, 1996, pp. 532-582. The Indian Pharmacopoeia, Ministry of Health and Family Welfare, Government of India, 1996, p. 188.
MANUSCRIPT Received 21 June 2004, accepted for publication 19 November 2004.
J. DRUG DEL. SCI. TECH. N° 3 Commission paritaire n° 0307T81401 - Dépôt légal 2e trim. 2005
252
Le directeur de la publication Dominique Dupont N° d’imprimeur : 45759 - Corlet Imprimeur SA 02 31 59 53 00
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal* Division of Pharmaceutics, Department of Pharmaceutical Technology, Jadavpur University, Jadavpur, Kolkata, PIN 700 032, India *Correspondence: [email protected] Ocular inserts containing ciprofloxacin hydrochloride (CPF) as a model drug were developed by optimization of design applying ANOVA. The parameters of optimization were taken as the concentration of calcium chloride solution and time of exposure of the treatment. Out of the four formulations studied, types III and IV contained hydroxypropylmethylcellulose (HPMC) whereas types I and II were developed without HPMC. Type I and III contained CPF as the soluble hydrochloride salt while in types II and IV CPF is present in crystal form. In vitro dissolution studies with the four formulations showed that time of 90% drug release for types II and IV were 23.8 ± 0.2 and 25.8 ± 2.6 h, respectively, whereas the same for types I and III were 8.9 ± 0.6 and 16.6 ± 1.5 h. In vivo studies on all the formulations in the rabbit eye revealed that tear concentrations of CPF lasted for more than 2 days with types II and IV inserts. All the formulations showed excellent retention properties in the cul-de-sac of rabbit eyes with almost no irritation. Key words: Ocular insert – Factorial design – Ciprofloxacin – Swelling index.
A significant challenge in drug delivery is the local administration of drugs to the eye [1]. The tendency of the eye to drain out instilled fluid volume in excess of 7 to 10 µl severely compromises the performance of liquid dosage forms such as solutions, suspensions and liposomes. Drugs from these dosage forms typically disappear from the cul-de-sac within 1 to 2 min of instillation in human beings and 5 to 10 min in rabbits [2, 3]. Ciprofloxacin hydrochloride (CPF) 0.3% w/v ophthalmic solution, one to two drops at a time, to the affected eye is administered to patients suffering from bacterial infections. Continuous medication for three consecutive days is required to reduce the bacterial conjunctivitis [4] and bacterial keratitis [5] in 95% of the patients. The in vitro minimum inhibitory concentration (MIC90) of CPF hydrochloride is 2 µg/ml [6] against all bacteria causing ocular infections. However, the treatment schedule described above gives a steady state concentration which is much above the MIC90 that may be uncomfortable to the patient, causing severe irritation due to its cationic nature. CPF also produces a white layer of crystals on the ulcerated portion of the cornea [5]. For these reasons, an ocular insert of CPF hydrochloride providing a concentration sufficiently above MIC90 in the tears, will have higher efficacy, prevent loss of drug and produce better patient compliance compared to eye drops. An attempt has been made in the present work to design an ocular insert that will maintain MIC90 of drug in the tears for as long as three days and also provide minimum swelling of the insert during its residence time in the cul-de-sac of the eye [7]. To achieve these objectives, CPF hydrochloride was incorporated in a hydrophilic matrix of sodium alginate film, cross-linked with calcium chloride to reduce swelling to the minimum and to provide prolonged release of the drug.
2. Methods
2.1. Preparation of ocular inserts Type I inserts were prepared by dissolving 25 mg of CPF hydrochloride and 25 mg of glycerol as plasticizer in 6 ml of distilled water, followed by dissolving 250 g of sodium alginate in the same solution. The homogeneous mass was cast on a stainless steel surface and then dried at 45°C for 8 h under an infrared heat source. The dried alginate films were removed and discs of 4.5 mm diameter were punched out with a single punching instrument. The discs were then treated with 1 to 4% w/v of calcium chloride solution for 15 to 60 s followed by blotting with filter paper to remove excess calcium chloride solution from the surface. The discs were finally air-dried for 1 h. For type II inserts, the solvent used to dissolve the drug and glycerol was a mixture of 5 ml of distilled water and 1 ml of Sorensenʼs phosphate buffer pH 7.4, which yielded a suspension of CPF, which was swirled with 250 mg sodium alginate to prepare a homogeneous, viscous mass. Type III inserts were prepared similar to type I but contained a suspension of 50 mg of HPMC and 200 mg sodium alginate dispersed in it. The type IV inserts were prepared in the same way as type II, but contained 50 mg of HPMC and 200 mg sodium alginate dispersed in it. The remaining steps for types II, III and IV are the same for type I. However, treatment with calcium chloride solution was done for type II, III and IV with 4% w/v solution and exposure time was only 15 s. 2.2. Experimental design In this study, a 22-factorial design [8] was adopted for type I inserts. The effects of two factors – (A) concentration of calcium chloride solution and (B) time of treatment – were studied at two levels: one at higher and the other at lower level since both controls the degree of cross-linking. Four experimental inserts were denoted as (I): 2% w/v CaCl2 concentration and 15 s time of exposure, a: 4% w/v and 15 s, b: 2% w/v and 60 s and ab: 4% w/v and 60 s. The area expansion index (α) of the inserts were measured. Swelling of the inserts in all dimensions should be minimum after prolonged contact to tears. The experiments were carried out in triplicate. The expansion indices were determined by placing one insert in each eye of albino rabbits weighing between 1.5 to 2.0 kg, and taking out after three days to observe their physical characteristics after bloating carefully. The inserts were scanned at 100 dpi (Canoscan N676U) against a black background and the images were pasted on a software made with
I. MATERIALS AND METHODS 1. Materials
Ciprofloxacin hydrochloride was obtained as a gift from Astra IDL, India. Sodium alginate, hydroxypropylmethylcellulose K4M, disodium hydrogen orthophosphate and sodium dihydrogen orthophosphate were purchased from Loba Chemie. Sorensenʼs modified isotonic phosphate buffer pH 7.4 was prepared and used wherever applicable because the rabbit lachrymal secretion has a pH of 7.4. In all experiments, glass double-distilled water was used. All other reagents and solvents were of analytical reagent grade. 249
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal
J. DRUG DEL. SCI. TECH., 15 (3) 249-252 2005
Visual Basic 6.0 programming language. The area of each insert was measured at the beginning and the end of study by checking the hue of each point that is different from the background color.
II. RESULTS AND DISCUSSION 1. Swelling studies of various treatment combinations of inserts
The extent of swelling of type I inserts in rabbit eyes, depicted in Table I, were measured from the projected diameter. It revealed that inserts treated with 4% calcium chloride solution produced less swelling than with 1% calcium chloride solution. The initial diameter of the inserts before treatment was 4.1 ± 0.03 mm. Just after treatment, the diameters of inserts obtained from treatment combination I showed the greatest swelling (4.72 ± 0.18 mm) while treatment combination ab produced minimum swelling (4.18 ± 0.08 mm). Both treatment combinations a and ab produced negligible and almost similar degree of swelling after treatment. Three days after remaining in contact with tears in the cul-de-sac of rabbit eyes, inserts with treatment combinations a and ab exhibited similar swelling indices and were sufficiently lower than treatment combinations I and b. The results of ANOVA resolves the variability of area expansion index into contributions due to two factors, i.e. concentration of calcium chloride and time of treatment. The contribution of each factor is measured having removed the effects of all other factors. Since the P value for the factor concentration of calcium chloride solution is less than 0.05, this factor has a statistically significant effect on area expansion index at a confidence level of 95%. Since treatments I and b produced inserts that were deformed after three days, the concentration of calcium chloride solution was taken at 4% w/v level instead of 1% w/v. Flexibility of the inserts were found to reduce if they were cured for 60 s at 4% w/v concentration of calcium chloride solution. So it was decided that the optimum levels of the factors were 4% w/v calcium chloride solution and exposure for 15 s, i.e. treatment combination a.
Area expansion index = [(final area - initial area)/initial area] x 100 Projected diameter dp = √(4 x area)/π Diameter expansion index = [(final dp - initial dp)/initial dp] x 100 2.3. Determination of drug content One insert was placed in 5 ml of 0.1 N sodium hydroxide solution to dissolve calcium alginate followed by neutralization with addition of 0.1 N hydrochloric acid. After filtration, the solution was assayed by UV spectrophotometer (Beckman DU-64) at 274 nm against a blank prepared similarly with an insert without drug. 2.4. In vitro drug release study The inserts were placed in 10 ml of Sorensenʼs modified isotonic phosphate buffer pH7.4 solution in 20-ml vials with rubber stopper, and then placed in an incubator, maintained at 37.0 ± 0.1°C. Onemillilitre sample was withdrawn and 1 ml of fresh phosphate buffer preheated to 37°C was replaced in the vial at the same time. Absorbances of the samples were measured using a Beckman DU-64 UV spectrophotometer at 274 nm after proper dilution against a blank of fresh phosphate buffer. 2.5. In vivo release study All animal experiments were conducted with prior approval of Institutional Animal Ethics Committee of Jadavpur University, Calcutta. Albino rabbits weighing between 1.5 to 2.0 kg, with no ocular pathology were selected for in vivo studies. In each eye, a sterile insert containing drug equivalent to 150 µg CPF hydrochloride was placed in the cul-de-sac. At appropriate time intervals of 1, 4, 8, 12, 24, 36, 48, 60 and 72 h, tear samples were collected by a capillary tube of internal diameter 383 µm having 1 µl mark lightly placed near the canthus of the eye without applying pressure. In case of conventional eye-drop, one drop of CPF hydrochloride 0.3% w/v ophthalmic solution was instilled in each eye with the help of a drop dispenser. Tear samples were collected at 0.25, 5, 10, 15, 30, 45 and 60 min after instillation. The concentration of CPF was determined by HPLC method [8] using a HPLC system (Perkin Elmer LC200) consisting of a series 200 pump and UV-Vis detector with a NCI 900 network chromatography interface and software (Total Chrome). The mixture of 87 volumes of 0.025 M phosphoric acid, previously adjusted with triethanolamine to a pH 3.0 ± 0.1 and 13 volumes of acetonitrile was used as the mobile phase at a flow rate of 1.5 ml/min and the detector wavelength at 278 nm.
2. In vitro drug release profile with treatment combination a
The release profile of drug from four types of inserts with treatment combination a are shown in Figure 1. Each experiment was performed in triplicate. In vitro parameters of four types of insert are depicted in Table II. Prolonged release of drug from type II and type IV inserts were evident from their time for 90% drug release, i.e. 23.8 ± 0.2 and 25.8 ± 2.6 h, respectively. Both contain crystals of CPF and were entrapped within the calcium alginate matrix. Dissolution of CPF from its crystals was the rate-limiting step because CPF has limited solubility in release medium of pH 7.4 buffer. The times for 90% drug release from type I and type III were much less compared to type II and IV inserts which appears due to the fact that they contain CPF in soluble hydrochloride form. However, in type III, higher entrapment of CPF hydrochloride was obtained due to the presence of HPMC that has possibly formed
Table I - Expansion indices of type I inserts after treatment and after three days in contact with tears. Parameters
(I)
a
Area before treatment (mm2) Projected diameter before treatment (mm) Area after treatment (mm2) Projected diameter after treatment (mm)
b
ab
16.90 ± 1.24
13.75 ± 0.54
13.40 ± 0.20* 4.13 ± 0.03** 17.53 ± 1.30
13.87 ± 0.70
4.72 ± 0.18
4.20 ± 0.11
4.64 ± 0.17
4.18 ± 0.08
Area after 3 days in contact with tear (mm2)
82.80 ± 14.48
37.76 ± 2.10
56.98 ± 14.44
36.12 ± 3.39
Projected diameter after 3 days in contact with tear (mm)
10.24 ± 0.90
6.93 ± 0.19
8.47 ± 1.12
6.78 ± 0.32
Area expansion index after 3 days in contact with tear (%)
372 ± 83
172 ± 15
237 ± 85
163 ± 25
Diameter expansion index after 3 days in contact with tear (%)
117 ± 19
65 ± 5
83 ± 24
62 ± 8
*Mean ± SD, n = 3. **Mean ± SD, n = 6. I, a, b and ab are treatment combinations. 250
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal
J. DRUG DEL. SCI. TECH., 15 (3) 249-252 2005
1400
100
Type-I Type-II Type-III
80
Type-IV
1000 Tear concentration (µg/ml)
% of drug released
1200
60 40 Type-I Type-II Type-III Type-IV
20
800
600
400
0 0
40
20
60
200
Time (h) 0
Figure 1 - Percentage of drug released versus time from type I, type II, type III and type IV inserts treated with 4% calcium chloride solution for 15 s. Mean ± SD, n = 3.
0
10
20
30
40
50
60
70
Time (h)
Figure 2 - Tear concentration (µg/ml) of CPF hydrochloride versus time profile after introducing inserts in the cul-de-sac of rabbit eyes. Mean ± SD, n = 3.
a gel structure in the calcium alginate matrix entrapping the dissolved drug molecules in it. The prolongation of drug release from type III was probably due to the gel structure of HPMC through which the movement of drug molecule was retarded compared to that from type I inserts. The linear regression coefficients of zero-order, Higuchi and firstorder models for drug release were calculated from data up to the time for 90% release. The regression coefficients showed that all types of insert correlate more with Higuchi (mean R = 0.9876) and first-order (mean R = 0.9828) models than with zero-order model (mean R = 0.9610).
completely disappearing after 30 min due to drainage and dilution with tears. Therefore, one drop is required every 15 min to maintain the drug level in tears. The in vivo release profiles of drug from all the four inserts were obtained and presented as tear concentration versus time in Figure 2. All experiments were conducted in triplicate and plotted as mean ± standard deviation. The pharmacokinetic parameters calculated from the in vivo data are represented in Table III. Tear concentration from all three inserts reached maximum value within 2.5 h, except type III which occurred in 11.3 ± 1.2 h. Type I inserts showed the highest peak tear concentration (1242.7 ± 99.4 µg/ml) among all the inserts followed by type IV (1040.3 ± 108.0 µg/ml). The quick release and high peak concentration of CPF in type I inserts were probably due to the fact that CPF was present in the dissolved salt state. The mean residence times (MRT) of drug in all four types of inserts were si-
3. In vivo drug release studies in the rabbit eye
Tear concentrations of CPF hydrochloride were measured for between 15 s and 60 min after instillation of one drop of the ophthalmic solution. The results revealed that the drug disappeared from the cul-de-sac within 30 min after instillation of the drop, the concentration of the drug reducing from 3,000 to 2,100 µg/ml within 15 s and Table II - In vitro parameters of various types of inserts (mean ± SD, n = 3). Parameters
Type I inserts
Type II inserts
Type III inserts
Type IV inserts
Drug content (µg/insert)
137.0 ± 9.2
131.3 ± 8.3
162.5 ± 7.2
129.3 ± 9.8
Time of 90% release (h)
8.9 ± 0.6
23.8 ± 0.2
16.6 ± 1.5
25.8 ± 2.6
0.9481 ± 0.0110
0.9706 ± 0.0057
0.9888 ± 0.0008
0.9366 ± 0.0436
R of zero-order model R of Higuchi model
0.9844 ± 0.0044
0.9968 ± 0.0021
0.9934 ± 0.0060
0.9757 ± 0.0144
R of first-order model
0.9788 ± 0.0168
0.9909 ± 0.0042
0.9831 ± 0.0160
0.9784 ± 0.0120
R of zero-order model = correlation coefficient between percentage of drug released and time. R of Higuchi model = correlation coefficient between percentage of drug released and square root of time. R of first-order model = correlation coefficient between logarithm of percentage of drug remaining to be released and time. Table III - Pharmacokinetic parameters after tear concentration (µg/ml) of CPF hydrochloride after introducing conventional drops and inserts in the cul-de-sac of rabbits. Parameters
Conventional CPF solution (0.3% w/v)
Type I inserts
Type II inserts
Type III inserts
Type IV inserts
Cmax (µg/ml)
-
1242.7 ± 99.4
680.7 ± 4.0
737.0 ± 836
1040.3 ± 108.0
Tmax (h)
-
2.7 ± 1.2
2.5 ± 0.9
11.3 ± 1.2
1.7 ± 0.6
MRT (h)
4.8 ± 0.5 min
8.5 ± 0.3
16.7 ± 0.5
12.9 ± 0.3
14.1 ± 0.8
Duration of action (h)
29.7 ± 1.2 min
38.1 ± 1.6
53.3 ± 0.6
44.0 ± 3.5
56.9 ± 6.9
Cmax = maximum concentration in tears. Tmax = time for the drug to reach Cmax. MRT = mean residence time = area under the first moment curve vs. time/area under the tear conc. curve vs. time. Duration of action = duration in which the concentration of drug remained above MIC90 (i.e. 2 µg/ml). 251
Design and optimization of ocular inserts for prolonged delivery of ciprofloxacin hydrochloride A. Samanta, S.K. Ghosal
J. DRUG DEL. SCI. TECH., 15 (3) 249-252 2005
gnificantly higher (P < 0.01) than conventional drops as depicted in Table III. Duration of action was assumed as the time for which the tear concentration remained above MIC90, i.e 2 µg/ml. It was found that conventional drops produced duration of action of only 29.7 min whereas durations of action were 38.1 and 44 h with type I and type III, respectively, and 53.3 and 56.9 h with types II and IV. Therefore, type II and type IV exhibited similar duration of action and a single insert is sufficient to produce the necessary therapeutic action for at least two days. Since the peak concentration is minimum (680.7 ± 4.0 µg/ml) with longer duration of action in type II inserts, it is thought to be the best formulation of the four types of insert studied. All of the ocular inserts showed excellent retention properties in the cul-de-sac of the rabbit with minimum swelling of the inserts. They did not come out even after three days. However, with the type II inserts the desirable properties were obtained with duration of action lasting for more than two days and not very high peak concentration of CPF, which might cause discomfort. Tear secretion and blinking rate in the rabbits were found to be normal after introducing the inserts, which indicates minimum or no irritation.
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PRAUSNIT M.R., NOONAN J.S.- Permeability of cornea, sclera and conjunctiva: A literature analysis for drug delivery to the eye.- J Pharm. Sci., 87, 1479-1488, 1998. LEE V.H.L., ROBINSON J.R.- Review: Topical ocular drug delivery: Recent developments and future challenges.- J. Ocular. Pharmacol., 2, 67-108, 1986. LEIBOWITZ H.M.- Antibacterial effectiveness of ciprofloxacin 0.3% ophthalmic solution in the treatment of bacterial conjunctivitis.- Am. J. Ophthalmol., 112, 295-335, 1991. LEIBOWITZ H.M.- Clinical evaluation of ciprofloxacin 0.3% ophthalmic solution for treatment of bacterial keratitis.- Am. J. Ophthalmol., 112, 345-475, 1991. DIAMOND J.P., WHITE L., LEEMING J.P., HOH H.B., EASTY D.L.- Topical 0.3% ciprofloxacin, norfloxacin, and ofloxacin in treatment of bacterial keratitis: a new method for comparative evaluation of ocular drug penetration.- Br. J. Opthalmol., 79, 606-609, 1995. DAROUGAR S., DAROUGAR D.- US Patent 6246971- East Croydon, GB. MONTGOMERY D.C. -The 2k factorial design.- In: Introduction to Statistical Quality Control, D.C. Montgomery Ed., John Wiley & Sons Inc., New York, 1996, pp. 532-582. The Indian Pharmacopoeia, Ministry of Health and Family Welfare, Government of India, 1996, p. 188.
MANUSCRIPT Received 21 June 2004, accepted for publication 19 November 2004.
J. DRUG DEL. SCI. TECH. N° 3 Commission paritaire n° 0307T81401 - Dépôt légal 2e trim. 2005
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