- Email: [email protected]
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action
J. DRUG DEL. SCI. TECH., 22 (6) 541-544 2012
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib1, M. El-Mahdy1, A. Abdel-Hafez2, S. Maher1* Faculty of Pharmacy, 2Faculty of Medicine, Assiut University, 71526, Assiut, Egypt *Correspondence: [email protected]
1
Microemulsions are promising drug delivery systems for ocular delivery of drugs, especially water insoluble drugs such as diclofenac in its acid undissociated form. Microemulsions are characterized with high surfactant content (> 10 % w/w) in order to lower the interfacial tension which facilitates dispersion process during the preparation of microemulsion and provides a flexible film around the droplets. However, there are a few researches that have studied the possible irritation effect of microemulsion on the eye. Therefore, evaluation of the ocular irritancy is an important requirement in the development of ocular delivery vehicles such as microemulsions. Draize test using rabbits was used for evaluation of the ocular irritation potential of the prepared microemulsions. The efficacy of the anti-inflammatory action of the formulation showing the least Draize score was then evaluated using 3 % croton oil in 2-ethoxyethanol to induce corneal inflammation in rabbits. Results showed that the tested formulations, M5 and M6, were non irritant (NI) where they showed a Draize score of 8 and 14 respectively. When M5 was studied for its anti-inflammatory action and compared with marketed eye drops, Epifenac, it showed a significantly shorter recovery time compared to Epifenac eye drops. Keywords: Microemulsion – Draize test – Ocular irritation – Croton oil – Anti-inflammatory – Diclofenac.
were evaluated. The characteristics of the prepared formulations were acceptable for application to the eye with regards to pH, particle size, and viscosity. Two formulations, M5 and M6, showed the highest in vitro release and so they should be assessed for further in vivo studies. This part of the work will focus on the study of any irritating and/ or damaging effects that might occur to the tissues of the eye after the instillation of the prepared formulations in order to ensure that the prepared formulations are safe for instillation into the eye. After that, the anti-inflammatory activity of formulations will be tested against a marketed dosage form (Epifenac eye drops) in order to find if a sustained release will be obtained. Tween 80 was used in preparing the tested microemulsions. Nonionic surfactants are the major type of surface active agents used in ophthalmic delivery systems since their advantages with respect to compatibility, stability, and toxicity are quite significant compared to the cationic, anionic, or amphoteric counterparts. They are generally less toxic, less hemolytic, and less irritating to the ocular surface, and tend to maintain pH near the physiological values (~7.4) when in solution [4]. Assessment of ocular irritation potential of ophthalmic products represents an extremely important step in their development. Since ophthalmic products are instilled in relatively high concentrations directly on the highly innervated corneal surface, it is not surprising that local irritation is sometimes encountered [5, 6]. The evaluation of ophthalmic products can be traced by the biological response to the drug through examination of the conjunctiva, cornea, and iris. For this purpose, rabbits are currently used to test the ocular toxicity and irritation of ophthalmic formulations. The rabbit has obvious advantages associated with its use. It is readily available, docile, easily handled, relatively inexpensive, easy to maintain, has large eyes, both corneal surface and bulbar conjunctival areas are large and easily observed, and the iris is unpigmented allowing ready observation to the iridal vessels. The focus of eye irritation testing is on assessing observable mucosal and epithelial effects. Systemic toxicity is not usually an outcome measure. Eye irritation is the constellation of reversible structural and inflammatory changes, whereas eye corrosion is irreversible damage that occurs to the ocular tissues following exposure to toxic material [7-9].
Microemulsions are considered an interesting alternative for conventional ophthalmic dosage forms. The industrial production and sterilization are relatively simple and inexpensive; they have good thermodynamic stability and inherently provide the capacity to make soluble lipophilic drugs, which depends also on the lipophilic phase used [1-2]. The preferential adsorption of the surfactant enables the modification of the physicochemical properties of the interface due to its amphiphilic nature. While the surfactant concentration is 0.1 % w/w in macroemulsions, it accounts for at least 10 % w/w in microemulsions due to the increase of the interface area between the aqueous and oily phases. This high concentration of surfactants can lead to ocular toxicity. That is why it might be better to decrease its quantity if possible [1]. In our previous work [3], six microemulsion formulations loaded with anti-inflammatory drug, diclofenac in its acidic form (water insoluble), were prepared using the constructed pseudo-ternary phase diagrams (Table I). Isopropyl myristate (IPM) was used as oil phase, Polyoxyethylene sorbitan monooleate (Tween 80) as a surfactant, glycerin as co-surfactant and isotonic Sörensen phosphate buffer (SPB) as aqueous phase. The aim of the work was to prepare ophthalmic microemulsions containing water insoluble drug (diclofenac) together with maintaining the convenience and simplicity of eye drops which might lead to a reduction of the frequency of application of the eye drops, resulting in improved patient compliance. The physicochemical characteristics (pH, viscosity, particle size and refractive index) of the prepared formulations together with stability and in vitro studies Table I - Compositions of the prepared microemulsion formulations (wt %) [3]. Formulation No.
IPM
SPB
Tween 80
Glycerin
Diclofenac
M1 M2 M3 M4 M5 M6
5 10 5 10 5 10
60 20 50 10 45 10
26.25 52.5 30 53.3 25 40
8.75 17.5 15 26.7 25 40
0.1 0.1 0.1 0.1 0.1 0.1 541
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib, M. El-Mahdy, A. Abdel-Hafez, S. Maher
J. DRUG DEL. SCI. TECH., 22 (6) 541-544 2012
I. Materials and methods 1. Materials
Diclofenac was a kind gift from Sedico (Egypt). Isopropyl myristate (IPM) was purchased from Merck Schichardt (Germany). Glycerin was purchased from Iso-Chem (Egypt), Polyoxyethylene sorbitan monooleate (Tween 80) was purchased from El Nasr Pharmaceutical Chemicals Co. Adwic (Egypt). Double distilled water was used in the preparation of Sörensen isotonic phosphate buffer (SPB), pH 7.4. Croton oil was purchased from Sigma Chemicals Co. (United States). All other ingredients were of pharmaceutical grade and were used as received.
Figure 1 - Inflammation scores obtained. Points (from right to left): 0, 1, 2, 3.
oil was used to induce corneal inflammation. This method permits evaluation of the topical anti-inflammatory activity of drug-loaded microemulsions[15, 16]. The inflammation score was observed visually according to modified Draize test [17]. Bulbar redness was evaluated. The score points range from 0 to 3 in which 0 is given for complete recovery while 3 is given for severe inflammation. The inflammatory reaction was induced by the instillation of 1 drop of 3 % croton oil in 2-ethoxyethanol in both eyes of the experimental animals. After instillation of croton oil, the bulbar redness score became 3 points in all animals [15]. After croton oil application, the inflammation score was 3 (Figure 1). One hour later, animals received 2 drops of the tested microemulsion. The treatment was applied daily till complete recovery (score 0 points). Eight rabbits (domestic and albino) of approximately 1.5-2.5 kg body weight were used. The right eye usually served as control or reference while the tested formulation was instilled into the left eye. The control eyes and the reference eyes received saline and the marketed eye drops Epifenac (containing diclofenac sodium), respectively. The onset of drug action and the time of recovery were recorded for each group. Results were expressed as mean value of eight readings ± standard deviation (SD). Statistical analysis was carried out employing two-way ANOVA tests. Difference at P < 0.05 was considered as a minimal level of significance using GraphPad Prism, Version 5.03.
2. Eye irritancy evaluation
Ocular tolerability, the potential ocular irritancy and/or damaging effects of primary selected formulations were evaluated according to a modified Draize test using a penlight [7]. Test procedures are carried out as follows: - each individual ingredient (IPM, Glycerin, and Tween 80) was tested using three rabbits, - the selected formulations, M5 and M6, (drug free) were tested on six rabbits. All tested rabbits were male rabbits (domestic) weighing 1.5-2.5 kg and free from any signs of ocular inflammation or gross abnormality. Cages should be designed to avoid accidental injury [10]. All studies were approved by the Ethics Committee at the Faculty of Pharmacy, Assiut University. In all the steps, rabbits were treated twice a day for 1 week. The right eye served as control and was treated with isotonic saline, while the tested materials were instilled into the left eye. Instillation of two drops (approximately 0.08 mL) is made into the lower conjunctival cul-de-sac; normal blinking is allowed, although the eyelids can be held together for several seconds after instillation [10]. The ocular condition was recorded every day after instillation and 1 h after the last administration using a slit-lamp [10]. The congestion, swelling, and discharge of the conjunctiva were graded according [7, 11] to a total score ranging from 0 to 110 according to Draize test, which is described in detail elsewhere [7]. A Draize irritation score of 15 points was used as the threshold. A material with an irritation score of 15 or more was classified as an irritant (I) and a chemical with an irritation score less than 15 was classified as a non-irritant (NI) [12, 13].
II. Results and discussion 1. Eye irritancy evaluation and histological test
When all ingredients (IPM, glycerin and Tween 80), with concentration 100 %, were tested individually, they gave total score (4) (zero for corneal and iris scores and 4 for conjunctival score) (Figure 2), in which vessels were definitely injected above normal and showed a slight amount of tearing. These results agree with Takahashi et al. [13]. M5 gave irritation score (8) (Figure 2), where they caused slight redness in the conjunctivae and a little tearing with moistening of the lids and hairs just adjacent to the lids and lasted for a few seconds after application of the eye drops. The rabbits also showed blinking a few times. Other than that, no changes in the eye had occurred. The eye in which the formulation was applied appeared similar to blank within 1 minute, with no irritation signs after 1 h. On the other hand, formulations M6 showed the highest irritation score (14) (Figure 2), where the conjunctivae became severely (beefy) red, slight swelling of the nictitating membrane and discharge which moistened the lids and a considerable area around the eye. Rabbits also
3. Histological test
After 1 week, animals which were used for testing the final formulation were sacrificed and eyes were isolated [14] and preserved in 10 % formalin for further histological tests [7]. The results were graded according to Table II.
4. In vivo study of the anti-inflammatory action on rabbits
After determining the irritation scores for the selected formulations, the ocular anti-inflammatory activity of the finally selected formulation (M5) was evaluated. In the present in vivo model in rabbits, croton Table II - Semiquantitative system grades the histopathological appearance of the corneal epithelium [7]. Assessment Normal surface epithelium with intact microvilli and tight junctions Some superficial cell sloughing and pitting with reduced microvilli Denuded superficial cells with intact underlying cells Partial loss of wing cells in the middle epithelial layers Loss of outermost epithelial cells exposing the basal epithelial cells
Score 0 1 2 3
Figure 2 - Different Draize irritation scores: 4, 8, 14, respectively, from left to right.
4 542
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib, M. El-Mahdy, A. Abdel-Hafez, S. Maher
J. DRUG DEL. SCI. TECH., 22 (6) 541-544 2012
showed blinking for a few times and the eye in which the formulation was applied appeared similar to blank within 1-4 min with no irritation signs after 1 h. The higher irritation caused by formulation M6 compared to M5 may be attributed to the high ratio of Tween 80 in M6 (40 % w/w) compared to that in M5 which is 25 % w/w. The histological examination of the eyes showed no change in their structures, in which the histological sections of both the control and the tested eyes are similar with no effect due to application of the tested formulations. The histopathological appearance grades of the corneal epithelium were 0 for all tested formulations which indicate that the applied formulations are safe to be applied without any fear of causing any reversible damage to the eye structure. From the irritation scores and the histological examination, the tested formulations can be considered non-irritant (NI) because their irritation scores are below 15 and so they can be applied safely to the eye. However, it is more convenient to use formulation M5 with the least score (8) for further in vivo studies on rabbits.
Table III - Time required for recovery of the inflamed eyes after application of M5 and Epifenac eye drops. Mean time (days) ± SD 2 to 1
Mean time (days) ± SD 1 to 0
Overall time for recovery 3 to 0 ± SD
M5
1.46 ± 0.42 ***
1.1 ± 0.32 ns
0.44 ± 0.18 ns
3 ± 0.6 ***
Epifenac
3.1 ± 0.6 **
1.25 ± 0.4 ns
0.88 ± 0.23 ns
5.2 ± 0.4 *
Control
3.8 ± 0.7
1.4 ± 0.5
1.125 ± 0.41
6.3 ± 0.5
M5
3,5
bulbar redness (points)
The anti-inflammatory effect of the tested formulation (M5) was explored by in vivo topical administration. This procedure is particularly adequate in processes which almost exclusively affect the anterior chamber of the eye in that the desired suppression of the inflammatory process is generally achieved with a lack of systemic secondary effects as a result of the treatment. A significant suppression of the inflammatory process induced by 3 % croton oil in 2-ethoxyethanol was recorded in each test performed using M5 and compared with the reference anti-inflammatory agent (Epifenac). The tested formulation significantly reduced bulbar redness score in comparison with Epifenac eye drops and control (Figure 1). The time required for recovery by M5 (3 ± 0.6 days) was significantly shorter in comparison with Epifenac eye drops (5.2 ± 0.4 days) (Table III, Figure 3). This may result due to the adsorption of the internal oil phase of diclofenac-loaded microemulsion (M5) on the corneal surface and, thus, providing a prolonged contact and release time. On the other hand, Epifenac eye drops were rapidly removed from the eye by tears and drainage through the nasolacrimal duct.
Epifenac eye drops
3
Control
2,5 2
1,5 1
0,5 0
0
2
4
6
8
Days Figure 3 - Time required for recovery of the inflamed eyes by different formulations (n = 8 ± SD). 3. 4. 5.
*
6.
Draize irritation test was carried out to evaluate the potential for ocular irritancy of microemulsion contacting IPM, Tween 80, glycerin and isotonic Sörensen phosphate buffer. Results showed that microemulsions caused no irritation to the eye which was then proved by histological studies. Formulation M5 can be instilled twice daily to the eye for the treatment of ocular inflammation after surgery or trauma gives significantly better and prolonged effects than water soluble diclofenac sodium in Epifenac. From the above mentioned description, it is recommended that, M5 should be subjected to further trials on experimental animals using different procedures for induction of ocular inflammation which can be then followed by clinical trials on patients.
7. 8.
9.
10.
References
2.
Mean time (days) ± SD 3 to 2
Each value is the average of 8 different experiments ± SD. *Significant, P < 0.05. **Moderately significant, P < 0.01. ***Highly significant , P < 0.0001.
2. In vivo study of the anti-inflammatory action on rabbits
1.
Formulation
11.
Vandamme T.F. - Microemulsions as ocular drug delivery systems: recent developments and future challenges. - Prog. Retin. Eye Res., 21, 15-34, 2002. Tenjarla S. - Microemulsions: an overview and pharmaceutical applications. - Crit. Rev. Ther. Drug Carrier Syst., 16, 62, 1999.
12. 13.
543
Habib F., El-Mahdy M., Maher S. - Microemulsions for ocular delivery: evaluation and characterization. - J. Drug Del. Sci. Tech., 21, 485-489, 2011. Remington J.P., Beringer P. - Interfacial phenomena. - In: Remington: The Science and Practice of Pharmacy, 21st ed., Philadelphia, Lippincott Williams & Wilkins, 2006, p. 280-292. Järvinen K., Järvinen T., Urtti A. - Ocular absorption following topical delivery. - Adv. Drug Deliv. Rev., 16, 3-19, 1995. Chetoni P., Burgalassi S., Monti D., Saettone M.F. - Ocular toxicity of some corneal penetration enhancers evaluated by electrophysiology measurements on isolated rabbit corneas. Toxicol. In vitro., 17, 497-504, 2003. Wilhelmus K.R. - The Draize eye test. - Surv. Ophthalmol., 45, 493-515, 2001. Abraham H.M., Kumarsingh R., Cometto-Muniz J.E., Cain W.S. - Draize eye scores and eye irritation thresholds in man combined into one quantitative structure-activity relationship. - Toxicol. In vitro., 12, 403-408, 1998. Burgalassi S., Chetoni P., Monti D., Saettone M.F. - Cytotoxicity of potential ocular permeation enhancers evaluated on rabbit and human corneal epithelial cell lines. - Toxicol. Lett., 122, 1-8, 2001. Gan L., Gan Y., Zhu C., Zhang X., Zhu J. - Novel microemulsion in situ electrolyte-triggered gelling system for ophthalmic delivery of lipophilic cyclosporine A: in vitro and in vivo results. - Int. J. Pharm., 365, 143-149, 2009. Agnihotri S.M., Vavia P.R. - Diclofenac-loaded biopolymeric nanosuspensions for ophthalmic application. - Nanomedicine, 5, 90-95, 2009. Kapoor Y., Howell B. A., Chauhan A. - Liposome assay for evaluating ocular toxicity of surfactants. - Invest. Ophthalmol. Vis. Sci., 50, 2727-2735, 2009. Takahashi Y., Koike M., Honda H., Ito Y., Sakaguchi H., Suzuki H., Nishiyama N. - Development of the short time exposure (STE)
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib, M. El-Mahdy, A. Abdel-Hafez, S. Maher
J. DRUG DEL. SCI. TECH., 22 (6) 541-544 2012
14.
15. 16.
test: an in vitro eye irritation test using SIRC cells. - Toxicol. In vitro., 22, 760-770, 2008. Cooper K.J., Earl L.K., Harbell J., Raabe H. - Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay. - Toxicol. In vitro., 15, 95-103, 2001. Villena C., Vivas J. M., Villar A.M. - Ocular inflammation models by topical application: Croton-oil induced uveitis. - Curr. Eye Res., 18, 3-9, 1999. Villena C., Vivas J.M., Villar A.M. - Suppression of croton oilinduced rabbit corneal edema by Sideritis javalambrensis. - J.
17.
Ethnopharmacol., 71, 301-305, 2000. Hardarson S.H., Sigurdsson H.H., Níelsdóttir G.E., Valgeirsson J., Loftsson T., Stefánsson E. - Ocular powder: dry topical formulations of timolol are well tolerated in rabbits. - J. Ocul. Pharmacol. Ther., 22, 340-346, 2006.
Manuscript Received 27 February 2012, accepted for publication 16 May 2012.
544
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib1, M. El-Mahdy1, A. Abdel-Hafez2, S. Maher1* Faculty of Pharmacy, 2Faculty of Medicine, Assiut University, 71526, Assiut, Egypt *Correspondence: [email protected]
1
Microemulsions are promising drug delivery systems for ocular delivery of drugs, especially water insoluble drugs such as diclofenac in its acid undissociated form. Microemulsions are characterized with high surfactant content (> 10 % w/w) in order to lower the interfacial tension which facilitates dispersion process during the preparation of microemulsion and provides a flexible film around the droplets. However, there are a few researches that have studied the possible irritation effect of microemulsion on the eye. Therefore, evaluation of the ocular irritancy is an important requirement in the development of ocular delivery vehicles such as microemulsions. Draize test using rabbits was used for evaluation of the ocular irritation potential of the prepared microemulsions. The efficacy of the anti-inflammatory action of the formulation showing the least Draize score was then evaluated using 3 % croton oil in 2-ethoxyethanol to induce corneal inflammation in rabbits. Results showed that the tested formulations, M5 and M6, were non irritant (NI) where they showed a Draize score of 8 and 14 respectively. When M5 was studied for its anti-inflammatory action and compared with marketed eye drops, Epifenac, it showed a significantly shorter recovery time compared to Epifenac eye drops. Keywords: Microemulsion – Draize test – Ocular irritation – Croton oil – Anti-inflammatory – Diclofenac.
were evaluated. The characteristics of the prepared formulations were acceptable for application to the eye with regards to pH, particle size, and viscosity. Two formulations, M5 and M6, showed the highest in vitro release and so they should be assessed for further in vivo studies. This part of the work will focus on the study of any irritating and/ or damaging effects that might occur to the tissues of the eye after the instillation of the prepared formulations in order to ensure that the prepared formulations are safe for instillation into the eye. After that, the anti-inflammatory activity of formulations will be tested against a marketed dosage form (Epifenac eye drops) in order to find if a sustained release will be obtained. Tween 80 was used in preparing the tested microemulsions. Nonionic surfactants are the major type of surface active agents used in ophthalmic delivery systems since their advantages with respect to compatibility, stability, and toxicity are quite significant compared to the cationic, anionic, or amphoteric counterparts. They are generally less toxic, less hemolytic, and less irritating to the ocular surface, and tend to maintain pH near the physiological values (~7.4) when in solution [4]. Assessment of ocular irritation potential of ophthalmic products represents an extremely important step in their development. Since ophthalmic products are instilled in relatively high concentrations directly on the highly innervated corneal surface, it is not surprising that local irritation is sometimes encountered [5, 6]. The evaluation of ophthalmic products can be traced by the biological response to the drug through examination of the conjunctiva, cornea, and iris. For this purpose, rabbits are currently used to test the ocular toxicity and irritation of ophthalmic formulations. The rabbit has obvious advantages associated with its use. It is readily available, docile, easily handled, relatively inexpensive, easy to maintain, has large eyes, both corneal surface and bulbar conjunctival areas are large and easily observed, and the iris is unpigmented allowing ready observation to the iridal vessels. The focus of eye irritation testing is on assessing observable mucosal and epithelial effects. Systemic toxicity is not usually an outcome measure. Eye irritation is the constellation of reversible structural and inflammatory changes, whereas eye corrosion is irreversible damage that occurs to the ocular tissues following exposure to toxic material [7-9].
Microemulsions are considered an interesting alternative for conventional ophthalmic dosage forms. The industrial production and sterilization are relatively simple and inexpensive; they have good thermodynamic stability and inherently provide the capacity to make soluble lipophilic drugs, which depends also on the lipophilic phase used [1-2]. The preferential adsorption of the surfactant enables the modification of the physicochemical properties of the interface due to its amphiphilic nature. While the surfactant concentration is 0.1 % w/w in macroemulsions, it accounts for at least 10 % w/w in microemulsions due to the increase of the interface area between the aqueous and oily phases. This high concentration of surfactants can lead to ocular toxicity. That is why it might be better to decrease its quantity if possible [1]. In our previous work [3], six microemulsion formulations loaded with anti-inflammatory drug, diclofenac in its acidic form (water insoluble), were prepared using the constructed pseudo-ternary phase diagrams (Table I). Isopropyl myristate (IPM) was used as oil phase, Polyoxyethylene sorbitan monooleate (Tween 80) as a surfactant, glycerin as co-surfactant and isotonic Sörensen phosphate buffer (SPB) as aqueous phase. The aim of the work was to prepare ophthalmic microemulsions containing water insoluble drug (diclofenac) together with maintaining the convenience and simplicity of eye drops which might lead to a reduction of the frequency of application of the eye drops, resulting in improved patient compliance. The physicochemical characteristics (pH, viscosity, particle size and refractive index) of the prepared formulations together with stability and in vitro studies Table I - Compositions of the prepared microemulsion formulations (wt %) [3]. Formulation No.
IPM
SPB
Tween 80
Glycerin
Diclofenac
M1 M2 M3 M4 M5 M6
5 10 5 10 5 10
60 20 50 10 45 10
26.25 52.5 30 53.3 25 40
8.75 17.5 15 26.7 25 40
0.1 0.1 0.1 0.1 0.1 0.1 541
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib, M. El-Mahdy, A. Abdel-Hafez, S. Maher
J. DRUG DEL. SCI. TECH., 22 (6) 541-544 2012
I. Materials and methods 1. Materials
Diclofenac was a kind gift from Sedico (Egypt). Isopropyl myristate (IPM) was purchased from Merck Schichardt (Germany). Glycerin was purchased from Iso-Chem (Egypt), Polyoxyethylene sorbitan monooleate (Tween 80) was purchased from El Nasr Pharmaceutical Chemicals Co. Adwic (Egypt). Double distilled water was used in the preparation of Sörensen isotonic phosphate buffer (SPB), pH 7.4. Croton oil was purchased from Sigma Chemicals Co. (United States). All other ingredients were of pharmaceutical grade and were used as received.
Figure 1 - Inflammation scores obtained. Points (from right to left): 0, 1, 2, 3.
oil was used to induce corneal inflammation. This method permits evaluation of the topical anti-inflammatory activity of drug-loaded microemulsions[15, 16]. The inflammation score was observed visually according to modified Draize test [17]. Bulbar redness was evaluated. The score points range from 0 to 3 in which 0 is given for complete recovery while 3 is given for severe inflammation. The inflammatory reaction was induced by the instillation of 1 drop of 3 % croton oil in 2-ethoxyethanol in both eyes of the experimental animals. After instillation of croton oil, the bulbar redness score became 3 points in all animals [15]. After croton oil application, the inflammation score was 3 (Figure 1). One hour later, animals received 2 drops of the tested microemulsion. The treatment was applied daily till complete recovery (score 0 points). Eight rabbits (domestic and albino) of approximately 1.5-2.5 kg body weight were used. The right eye usually served as control or reference while the tested formulation was instilled into the left eye. The control eyes and the reference eyes received saline and the marketed eye drops Epifenac (containing diclofenac sodium), respectively. The onset of drug action and the time of recovery were recorded for each group. Results were expressed as mean value of eight readings ± standard deviation (SD). Statistical analysis was carried out employing two-way ANOVA tests. Difference at P < 0.05 was considered as a minimal level of significance using GraphPad Prism, Version 5.03.
2. Eye irritancy evaluation
Ocular tolerability, the potential ocular irritancy and/or damaging effects of primary selected formulations were evaluated according to a modified Draize test using a penlight [7]. Test procedures are carried out as follows: - each individual ingredient (IPM, Glycerin, and Tween 80) was tested using three rabbits, - the selected formulations, M5 and M6, (drug free) were tested on six rabbits. All tested rabbits were male rabbits (domestic) weighing 1.5-2.5 kg and free from any signs of ocular inflammation or gross abnormality. Cages should be designed to avoid accidental injury [10]. All studies were approved by the Ethics Committee at the Faculty of Pharmacy, Assiut University. In all the steps, rabbits were treated twice a day for 1 week. The right eye served as control and was treated with isotonic saline, while the tested materials were instilled into the left eye. Instillation of two drops (approximately 0.08 mL) is made into the lower conjunctival cul-de-sac; normal blinking is allowed, although the eyelids can be held together for several seconds after instillation [10]. The ocular condition was recorded every day after instillation and 1 h after the last administration using a slit-lamp [10]. The congestion, swelling, and discharge of the conjunctiva were graded according [7, 11] to a total score ranging from 0 to 110 according to Draize test, which is described in detail elsewhere [7]. A Draize irritation score of 15 points was used as the threshold. A material with an irritation score of 15 or more was classified as an irritant (I) and a chemical with an irritation score less than 15 was classified as a non-irritant (NI) [12, 13].
II. Results and discussion 1. Eye irritancy evaluation and histological test
When all ingredients (IPM, glycerin and Tween 80), with concentration 100 %, were tested individually, they gave total score (4) (zero for corneal and iris scores and 4 for conjunctival score) (Figure 2), in which vessels were definitely injected above normal and showed a slight amount of tearing. These results agree with Takahashi et al. [13]. M5 gave irritation score (8) (Figure 2), where they caused slight redness in the conjunctivae and a little tearing with moistening of the lids and hairs just adjacent to the lids and lasted for a few seconds after application of the eye drops. The rabbits also showed blinking a few times. Other than that, no changes in the eye had occurred. The eye in which the formulation was applied appeared similar to blank within 1 minute, with no irritation signs after 1 h. On the other hand, formulations M6 showed the highest irritation score (14) (Figure 2), where the conjunctivae became severely (beefy) red, slight swelling of the nictitating membrane and discharge which moistened the lids and a considerable area around the eye. Rabbits also
3. Histological test
After 1 week, animals which were used for testing the final formulation were sacrificed and eyes were isolated [14] and preserved in 10 % formalin for further histological tests [7]. The results were graded according to Table II.
4. In vivo study of the anti-inflammatory action on rabbits
After determining the irritation scores for the selected formulations, the ocular anti-inflammatory activity of the finally selected formulation (M5) was evaluated. In the present in vivo model in rabbits, croton Table II - Semiquantitative system grades the histopathological appearance of the corneal epithelium [7]. Assessment Normal surface epithelium with intact microvilli and tight junctions Some superficial cell sloughing and pitting with reduced microvilli Denuded superficial cells with intact underlying cells Partial loss of wing cells in the middle epithelial layers Loss of outermost epithelial cells exposing the basal epithelial cells
Score 0 1 2 3
Figure 2 - Different Draize irritation scores: 4, 8, 14, respectively, from left to right.
4 542
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib, M. El-Mahdy, A. Abdel-Hafez, S. Maher
J. DRUG DEL. SCI. TECH., 22 (6) 541-544 2012
showed blinking for a few times and the eye in which the formulation was applied appeared similar to blank within 1-4 min with no irritation signs after 1 h. The higher irritation caused by formulation M6 compared to M5 may be attributed to the high ratio of Tween 80 in M6 (40 % w/w) compared to that in M5 which is 25 % w/w. The histological examination of the eyes showed no change in their structures, in which the histological sections of both the control and the tested eyes are similar with no effect due to application of the tested formulations. The histopathological appearance grades of the corneal epithelium were 0 for all tested formulations which indicate that the applied formulations are safe to be applied without any fear of causing any reversible damage to the eye structure. From the irritation scores and the histological examination, the tested formulations can be considered non-irritant (NI) because their irritation scores are below 15 and so they can be applied safely to the eye. However, it is more convenient to use formulation M5 with the least score (8) for further in vivo studies on rabbits.
Table III - Time required for recovery of the inflamed eyes after application of M5 and Epifenac eye drops. Mean time (days) ± SD 2 to 1
Mean time (days) ± SD 1 to 0
Overall time for recovery 3 to 0 ± SD
M5
1.46 ± 0.42 ***
1.1 ± 0.32 ns
0.44 ± 0.18 ns
3 ± 0.6 ***
Epifenac
3.1 ± 0.6 **
1.25 ± 0.4 ns
0.88 ± 0.23 ns
5.2 ± 0.4 *
Control
3.8 ± 0.7
1.4 ± 0.5
1.125 ± 0.41
6.3 ± 0.5
M5
3,5
bulbar redness (points)
The anti-inflammatory effect of the tested formulation (M5) was explored by in vivo topical administration. This procedure is particularly adequate in processes which almost exclusively affect the anterior chamber of the eye in that the desired suppression of the inflammatory process is generally achieved with a lack of systemic secondary effects as a result of the treatment. A significant suppression of the inflammatory process induced by 3 % croton oil in 2-ethoxyethanol was recorded in each test performed using M5 and compared with the reference anti-inflammatory agent (Epifenac). The tested formulation significantly reduced bulbar redness score in comparison with Epifenac eye drops and control (Figure 1). The time required for recovery by M5 (3 ± 0.6 days) was significantly shorter in comparison with Epifenac eye drops (5.2 ± 0.4 days) (Table III, Figure 3). This may result due to the adsorption of the internal oil phase of diclofenac-loaded microemulsion (M5) on the corneal surface and, thus, providing a prolonged contact and release time. On the other hand, Epifenac eye drops were rapidly removed from the eye by tears and drainage through the nasolacrimal duct.
Epifenac eye drops
3
Control
2,5 2
1,5 1
0,5 0
0
2
4
6
8
Days Figure 3 - Time required for recovery of the inflamed eyes by different formulations (n = 8 ± SD). 3. 4. 5.
*
6.
Draize irritation test was carried out to evaluate the potential for ocular irritancy of microemulsion contacting IPM, Tween 80, glycerin and isotonic Sörensen phosphate buffer. Results showed that microemulsions caused no irritation to the eye which was then proved by histological studies. Formulation M5 can be instilled twice daily to the eye for the treatment of ocular inflammation after surgery or trauma gives significantly better and prolonged effects than water soluble diclofenac sodium in Epifenac. From the above mentioned description, it is recommended that, M5 should be subjected to further trials on experimental animals using different procedures for induction of ocular inflammation which can be then followed by clinical trials on patients.
7. 8.
9.
10.
References
2.
Mean time (days) ± SD 3 to 2
Each value is the average of 8 different experiments ± SD. *Significant, P < 0.05. **Moderately significant, P < 0.01. ***Highly significant , P < 0.0001.
2. In vivo study of the anti-inflammatory action on rabbits
1.
Formulation
11.
Vandamme T.F. - Microemulsions as ocular drug delivery systems: recent developments and future challenges. - Prog. Retin. Eye Res., 21, 15-34, 2002. Tenjarla S. - Microemulsions: an overview and pharmaceutical applications. - Crit. Rev. Ther. Drug Carrier Syst., 16, 62, 1999.
12. 13.
543
Habib F., El-Mahdy M., Maher S. - Microemulsions for ocular delivery: evaluation and characterization. - J. Drug Del. Sci. Tech., 21, 485-489, 2011. Remington J.P., Beringer P. - Interfacial phenomena. - In: Remington: The Science and Practice of Pharmacy, 21st ed., Philadelphia, Lippincott Williams & Wilkins, 2006, p. 280-292. Järvinen K., Järvinen T., Urtti A. - Ocular absorption following topical delivery. - Adv. Drug Deliv. Rev., 16, 3-19, 1995. Chetoni P., Burgalassi S., Monti D., Saettone M.F. - Ocular toxicity of some corneal penetration enhancers evaluated by electrophysiology measurements on isolated rabbit corneas. Toxicol. In vitro., 17, 497-504, 2003. Wilhelmus K.R. - The Draize eye test. - Surv. Ophthalmol., 45, 493-515, 2001. Abraham H.M., Kumarsingh R., Cometto-Muniz J.E., Cain W.S. - Draize eye scores and eye irritation thresholds in man combined into one quantitative structure-activity relationship. - Toxicol. In vitro., 12, 403-408, 1998. Burgalassi S., Chetoni P., Monti D., Saettone M.F. - Cytotoxicity of potential ocular permeation enhancers evaluated on rabbit and human corneal epithelial cell lines. - Toxicol. Lett., 122, 1-8, 2001. Gan L., Gan Y., Zhu C., Zhang X., Zhu J. - Novel microemulsion in situ electrolyte-triggered gelling system for ophthalmic delivery of lipophilic cyclosporine A: in vitro and in vivo results. - Int. J. Pharm., 365, 143-149, 2009. Agnihotri S.M., Vavia P.R. - Diclofenac-loaded biopolymeric nanosuspensions for ophthalmic application. - Nanomedicine, 5, 90-95, 2009. Kapoor Y., Howell B. A., Chauhan A. - Liposome assay for evaluating ocular toxicity of surfactants. - Invest. Ophthalmol. Vis. Sci., 50, 2727-2735, 2009. Takahashi Y., Koike M., Honda H., Ito Y., Sakaguchi H., Suzuki H., Nishiyama N. - Development of the short time exposure (STE)
Microemulsion for ocular delivery: ocular irritancy test and in vivo studies of anti-inflammatory action F. Habib, M. El-Mahdy, A. Abdel-Hafez, S. Maher
J. DRUG DEL. SCI. TECH., 22 (6) 541-544 2012
14.
15. 16.
test: an in vitro eye irritation test using SIRC cells. - Toxicol. In vitro., 22, 760-770, 2008. Cooper K.J., Earl L.K., Harbell J., Raabe H. - Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay. - Toxicol. In vitro., 15, 95-103, 2001. Villena C., Vivas J. M., Villar A.M. - Ocular inflammation models by topical application: Croton-oil induced uveitis. - Curr. Eye Res., 18, 3-9, 1999. Villena C., Vivas J.M., Villar A.M. - Suppression of croton oilinduced rabbit corneal edema by Sideritis javalambrensis. - J.
17.
Ethnopharmacol., 71, 301-305, 2000. Hardarson S.H., Sigurdsson H.H., Níelsdóttir G.E., Valgeirsson J., Loftsson T., Stefánsson E. - Ocular powder: dry topical formulations of timolol are well tolerated in rabbits. - J. Ocul. Pharmacol. Ther., 22, 340-346, 2006.
Manuscript Received 27 February 2012, accepted for publication 16 May 2012.
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