http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–7 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.861883
Formulation and evaluation of Cyclosporin A emulgel for ocular delivery Yan Shen1, Xiang Ling1, Weiwei Jiang2, Shuang Du1, Yang Lu3, and Jiasheng Tu1 Department of Pharmaceutics, China Pharmaceutical University, Nanjing, China, 2Patent Examination Cooperation Center of SIPO, Jiangsu, Suzhou, China, and 3Department of Industrial Pharmacy, School of Chinese Pharmacy, Beijing University of TCM, Beijing, China
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
1
Abstract
Keywords
Emulgels have been extensively covered as a promising drug delivery system for the administration of lipophilic drugs. This work was conducted to develop an emulgel formulation for Cyclosporin A (CsA) employing polycarbophil as the gelling agent for ocular delivery. The prepared emulgels were evaluated for their physical appearance, rheological behavior, drug release, stability, precorneal clearance and irritation. Results showed that CsA emulgel formulations prepared with polycarbophil exhibited acceptable physical properties and drug release, which remained consistent after storage for 3 months. A prolonged retention time was also observed on the ocular surface with improved ocular bioavailability and no irritation. Therefore, the polycarbophil-based emulgel could be exploited as a potential hydrophobic drug carrier for topical ocular drug delivery.
Cyclosporin A, emulgel, ocular delivery, polycarbophil, precorneal clearance
Introduction A gel is a jelly-like material with a three-dimensional crosslinked network of colloidal solid particles within which miraculous amounts of aqueous or hydro alcoholic liquid could be entrapped (Kumar & Verma, 2011). It is superior in terms of the application as a dosage form and patient acceptability. Despite many advantages, a major obstacle for gels is in the delivery of hydrophobic drugs. So to conquer this impediment, emulgels have emerged as one of the most interesting controlled release drug delivery systems which possess the properties of both emulsions and gels (Steiger, 2010). In the past decade, scientists and industrial researchers have taken increasing interests in the field of pharmaceutical semisolid dosage forms especially emulgels, primarily due to their homogenous behavior and jelly-like consistency. Emulgels, also known as o/w emulsion gels or cream gels, are colloidal systems mixed with oil-in-water solutions as an interior phase. Despite their utilization in cosmetics for a long period, wide application of such hydrophilic systems in pharmaceutics starts with dermatological formulations (Marquardt & Sucker, 1998). In recent years, discovery of novel polymers with diverse functions of improved gelling capacity and as emulsifiers and thickeners dramatically facilitated the development of emulgels; which decreased superficial and interfacial tension, and increased the viscosity
Address for correspondence: Yan Shen, Department of Pharmaceutics, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China. Tel: 0086-25-83271305. Fax: 0086-25-83271351. Email: shenyan [email protected] Yang Lu, Department of Industrial Pharmacy, School of Chinese Pharmacy, Beijing University of TCM, 6 Zhong Huan Nan Lu, Beijing 100102, China. Email: [email protected]
History Received 27 August 2013 Revised 30 October 2013 Accepted 30 October 2013
of the aqueous phase, resulting in stable emulsions and creams (Gupta et al., 2010). Eventually, their rheological properties can be controlled easily by the proper selection of the gelling agent as well as the oily ingredient. CsA, which has a selective immunosuppressive effect, is used for several autoimmune as well as contingent eye diseases for its specific inhibition on the T-cell-dependent immune reactions at high-rate (Belin et al., 1990; Schreiber & Crabtree, 1992; Schumacher & Nordheim, 1992). CsA is a pretty hydrophobic drug with poor water solubility (0.012 mg/mL at 25 C) (Mondon et al., 2011), hence, creating formulation problems. To yield an aqueous CsA formulation, many approaches have been attempted such as co-solvent, pro-drugs, preparation in different oils (e.g. castor, olive or peanut) or emulsions (Stevenson et al., 2000; Chen et al., 2012; Rodriguez-Aller et al., 2012). Nevertheless, the use of these approaches is generally limited by the appearance of side-effects which may include itching, redness, burning and toxic effects on the cornea (Benitez del Castillo et al., 1994; Lallemand et al., 2003). An eye drop solution is the best choice for topical therapy of ocular diseases, notably, in some cases, when a localized action (e.g., the cornea and/or anterior chamber) of drug is needed. However, self-protecting mechanisms, such as lacrimal secretion and blinking reflex, along with corneal impermeability, make drug retention time so short that frequent administration is necessary for highly efficient therapy (Wei et al., 2002). In order to avoid the rapid dilution, formulations with an increased viscosity have been investigated. The increase of both the residence time of drug in the precorneal area and the absorption can improve its therapeutic effect. Recently, Durasite (Insite Vision, Inc.), a polycarbophilbased optical formulation (polycarbophil 0.9%) with
2
Y. Shen et al.
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
azithromycin 1.0% (Azasite) and besifloxacin 0.6% (Besivance) for the treatment of bacterial conjunctivitis, was introduced into the market. In addition, after a long-term topical ophthalmic administration on rabbits, the polycarbophil-based formulation was proved to be harmless for chronic use as well as impressive in improving the residence time of drug on the ocular surface, thus a promising ophthalmic medicinal preparation for the treatment of eye diseases (Krenzer et al., 2012). Based on these considerations and our preliminary studies, the aim of this work was to develop an emulgel formulation of CsA using polycarbophil as gelling agent. The physical, rheological characteristic, stability under several conditions, in vitro release, eye irritation studies, and the residence time of the drug in the precorneal area of the prepared emulgels were all evaluated.
Materials and methods Materials and animals Cyclosporine A (CsA) was purchased from Galena (Czech Republic). Polycarbophil was obtained from The Lubrizol Corporation (Ohio Clif Lancaster, USA). Poloxamer188 was supplied by Nanjing Well Chemical Co., Ltd (Nanjing, China). Castor oil (analytical reagent) was purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Dialysis membrane was purchased from Sigma Chemical Company (Shanghai, China). Methanol and acetonitrile were of HPLC grade and other chemicals were all of analytical reagent grade. Rabbits were purchased from the Experimental Animal Center of Nanjing Qinglongshan (Nanjing, China). The animals were acclimatized at 25 1 C and 70 5% relative humidity under natural light/dark conditions for 1 week before experiments. All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of China Pharmaceutical University.
Drug Deliv, Early Online: 1–7
to the sample was performed four times using a Sonicator B12 (Branson Sonic Power Company, Danbury, CT). After each sonication, the dispersion was mixed manually. At last, the emulgel system was jellified by adding different volume ratios of the CsA O/W emulsion and the polycarbophil dispersion was then sheared at 200 rpm for 45 min. The pH of the emulgel was regularly adjusted to 6.8 using NaOH or HCl (Mohamed, 2004; Shahin et al., 2011) to maintain its viscosity. The product of emulgel was formulated by now. The formulations were repeated for three times. Evaluation of emulgel Physical examination. The prepared emulgel formulations
were examined for their physical characteristics including color, homogeneity and phase separation. pH measurements: pH was measured at room temperature using a Scientific Instruments model IQ150 pH meter with a PH17-SS pH electrode (Scientific Instruments, Inc., San Diego, California, USA). Osmolarity measurements: Freezing point depression method (Micro-Osmometer Model 3300, Advanced Instruments, Norwood, MA) was used to obtain osmotic pressure (P) versus osmolality curve for CsA. All measurements were done in duplicate.
Methods
Viscosity determination of the prepared formulations was carried out on a cone (0.8 ) and plate geometry viscometer (Brookfield, USA) using spindle SC4-16. Viscosity of sample solutions was measured at different angular velocities at 37 1 C. A typical run comprised changing the angular velocity from 25 to 220 rpm at a controlled ramp speed. After 6 s at 25 rpm, the velocity was increased to 220 rpm with a similar wait time at each speed. The hierarchy of angular velocity was reversed (from 220 to 25 rpm) with a similar wait time of 6 s. Eventually, the average of two readings was used to calculate the viscosity. Evaluations were conducted in triplicate (Perioli et al., 2008).
Preparation of emulgels
Stability studies
The CsA cannot be easily dissolved in castor oil, in order to obtain the O/W emulsion, 100 mg CsA was added to 1 ml ethanol and 1.5 g castor oil was then added to form a homogeneous oil phase. After evaporation of ethanol under vacuum with a rotary evaporator, the oil phase of CsA dissolved in castor oil was obtained. Afterwards, the aqueous phase was prepared by dissolving 330 mg poloxamer 188 in ultrapure water. In the next step, both oil and aqueous phase were mixed together under continuous magnetic stirring at room temperature. Finally, the O/W emulsion was formed with a homogenizer (Erweka, type AR 401, Heusenstamm, Germany) for 5 min at 10 000 rpm. For the purpose of preparing the polycarbophil dispersion, 0.2 g of the polymer powder was dispersed in an iso-osmotic solution containing 3.0% (w/v) glycerol. pH was adjusted to physiological pH 6.8 by adding the required amount of NaOH. To ensure complete hydration, the solutions were stored at 6 C for a minimum of 12 h. And then, an additional sonication procedure of 30 s by imposing 80 W power output
The prepared CsA emulgel formulations were stored away from light in high-density polyethylene bottles at 40 C and 4 C for 3 months, respectively. Samples were withdrawn at 15-day time intervals and evaluated for their physical appearance, pH, osmotic pressure, rheological behavior and drug content (Niazi, 2009).
Rheological study.
Drug release study in simulated lachrymal fluid Simulated lachrymal fluid (SLF) is an electrolyte solution containing 1.7893 g/L KCl, 6.3118 g/L NaCl, 2.1842 g/L NaHCO3, 44.4 mg/L CaCl2 and 47.6 mg/L MgCl2 (Van Haeringen, 1981). Physiological pH (7.4 0.1) was adjusted by adding the required amount of 0.1 mol/L HCl. Due to the poor solubility of CsA in the water, the SLF containing 30% ethanol was used as the release medium. CsA emulgel release kinetics were described by using a dialysis-bag method (Er et al., 2009). In brief, the CsA emulgel (1 g) was added to the dialysis bag (cut-off
Formulation and evaluation of CsA emulgel for ocular delivery
DOI: 10.3109/10717544.2013.861883
weight: 3500 Da) and then placed at the bottom of the dissolution meter (100 rpm) at 34 C. The mount of the drug adsorbed by the dialysis bag was negligible. Each dissolution cup was added with 30 mL release medium. After that, the amount of CsA released from the emulgel was determined by HPLC at predetermined time points. 2.0 mL sample was withdrawn from the dissolution cup, and an equal volume of the release medium was refilled to maintain the original volume of the dissolution cup. Each experiment was run six times. CsA release was expressed as below (Equation 1) and plotted as a function of time: CsA release % ¼
Amount of CsA released to the medium Total amount of CsA in the dialysis bag 100%
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
ð1Þ Baseline studies on free CsA solutions have shown that free drug transport through the dialysis membrane is fast, in comparison to that released from emulgel while the nonspecific free CsA adsorption on the dialysis membrane was negligible. The in vitro release profiles of CsA emulgels were compared using similarity factors, f2, as defined by the following Equation (2) (Costa, 2001): ( ) 0:5 1 Xn 2 f2 ¼ 50 log 1 þ ðR Tt Þ 100 ð2Þ t1 t n where n is number of time points, and Tt and Rt are percentage releases at time point (t) for the reference and test formulation, respectively. Cyclosporine A quantification CsA concentrations were determined by the HPLC method. The HPLC system employed in the study consisted of an Agilent G1100 HPLC series system (Agilent Technologies, Palo Alto, CA) equipped with an Agilent G1310A pump and an Agilent G1314A variable wavelength UV-VIS photo diode array detector (PDAD) set at 210 nm. A Waters Xbridge-C18 ODS column (150 mm 4.6 mm, 5 mm) was operated at 70 C due to the existence of various conformers of CsA. The mobile phase consisted of acetonitrile–water–methanol–phosphoric acid (650:300:50:0.4, v/v/v/v) was flowing at a rate of 0.6 mL/min. A calibration curve was established on each running day. These curves showed good linearity, with correlation coefficients no less than 0.99. The limit of quantification (LOQ) and detection (LOD) were 50 and 20 ng/mL, respectively. Precorneal clearance study Ten rabbits divided into two groups (five rabbits in each group) were used to determine precorneal clearance. For emulgel group, each rabbit was instilled with 50 mL CsA emulgel preparation according to the section ‘‘Preparation of emulgels’’ (0.05%) on the right eye, and 50 mL blank saline solution on the left eye, respectively. For castor oil group, each rabbit was instilled with 50 mL CsA castor oil ophthalmic solution (0.05%) on the right eye, and 50 mL blank saline solution on the left eye, respectively. At 0, 0.5, 1, 2, 4, 7, 14,
3
24 h after instillation, tear samples of both eyes were collected by Schirmer test strips. The amount of tear withdrawn was calculated by subtracting the weight of each strip after sampling from the weight before sampling. Subsequently, the Schirmer strip was placed into an Eppendorf tube, dried under N2 stream, and then 0.2 mL of mobile phase was added. The sample was vortexed for 30 s thoroughly to dissolve CsA into the mobile phase and centrifuged under 300 g. All the experiment were performed under the Protocol of Ethical Comittee Permission. Pharmacokinetic and statistical analysis Pharmacokinetic analysis was accomplished using the 3p87 Pharmacokinetic Program (Chinese Society of Mathematical Pharmacology 1987, China). Statistical comparisons were performed using analysis of variance (ANOVA) or the Student’s t-test, where appropriate, and statistical significance was set at p50.05. In vivo eye irritation assessment in rabbits None of the rabbits exhibited ocular defects or pre-existing corneal injury in the pre-experimental inspection using an ophthalmoscope 24 h prior to administration. After rabbits were randomly divided into two groups with five in each, approximately 50 mL of CsA emulgel (0.05%) and 0.9% saline solution was dropped into the conjunctival sac of the left eye of each animal. The lids were then gently held together for about 1 s in order to prevent loss of the material. The right eye, which remained untreated, served as control. Animals wore Elizabethan collars during the study to avoid licking and scratching their eyes and faces. The rabbits’ eyes were instilled on day 1, 2, 3, 4 and 7. Then residues of the prepared formulations were removed from eyes by washing with 0.9% saline solution at 168 h (7 days) post-administration (Kishore et al., 2009). The animals were then euthanized by air embolism after being deeply anesthetized with an intramuscular overdose of anesthetic and paralyzing mixture of xylazine (20 mg/kg) and ketamine (200 mg/kg). Eyeball and lid tissues were removed and fixed in Davison’s fixative solution composed of 1 part glacial acetic acid, 2 parts 37% formaldehyde, 3 parts 95% ethanol, and 3 parts distilled water. Following fixation, they were embedded in paraffin for pathology. Eyeballs were determined in a masked fashion on the basis of following criteria: variation of the ocular surface epithelial cells, edema in lid tissues, presence of inflammatory cells, and any other abnormality (Diebold et al., 2007).
Results and discussion Physical appearance The prepared CsA emulgel formulations were white viscous creamy preparations with smooth and homogeneous appearance. They were easily droppable with acceptable viscosity and fair mechanical properties. The pH value of all the prepared formulations ranged from 6.3 to 6.5 and the osmotic pressure values ranged from 245 to 310 Osmol/kg, which are considered acceptable to avoid the risk of irritation upon application to the eye.
4
Y. Shen et al.
Drug Deliv, Early Online: 1–7
Table 1. The stability of CsA emulgel. Month
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Content Viscosity (cP) pH Osmotic pressure (mOsmol/kg) Appearance
0
1
3
99.85% 1888 6.66 301
99.57% 1917 6.67 308
99.63% 1924 6.66 303
Milk white, not layed
Milk white, not layed
Milk white, not layed
Figure 1. Viscosity-shear rate profiles of CsA emulgel.
Rheological studies The tests were performed at a rotating speed ranging from 25 to 220 rpm for 20 min. Results are shown in Figure 1. The formulations exhibited pseudo-plastic rheology as evinced by shear thinning and an increase in the shear stress with enhanced angular velocity, which are consistent with our preliminary data obtained during formulation screening (Du et al., 2011). In this flow behavior, static molecules could be entrapped in the cross-linked network. When sheared to decrease the flow resistance, the molecules would be realigned along the direction of flow, thus resulting in the lower viscosity (Parrott, 1970). The viscosity of the formulations prepared was (2312 25) cp with SC16 rotor at 50 rpm. Shear thinning behavior is a desirable property of topical ophthalmic preparations since it should thin during application and thicken otherwise (Van Ooteghem, 1993). Since the ocular shear rate is very high, ranging from 0.03 s1 during inter-blinking periods to 4250–28 500 s1 during blinking (Bother & Waaler, 1990), viscoelastic fluids with a viscosity that is high under the low shear rate conditions and low under the high shear rate conditions are often preferred, which could spread easily on the ocular surface and then increase the ocular bioavailability as well as the compliance of patients.
Figure 2. The effects of concentration and viscosity of gel on the release behavior of CsA emulgel.
Stability study The physical appearance, pH, osmotic pressure, rheological properties and drug content were investigated to evaluate the stability of the prepared formulations, and no significant changes were observed (Table 1). The results showed that all emulgel formulations stored for up to 3 months were as stable as the newly prepared ones. In vitro release study To make a deep comparison, we investigated the in vitro release behaviors of three emulgel formulations with varying polycarbophil contents of 0.2%, 0.25% and 0.3%. As shown in Figure 2, the 0.2% polycarbophil emulgel formulation with the lowest viscosity was observed to exhibit the best drug
Figure 3. Concentration–time profiles of CsA in tear fluid after instillation in conscious rabbits (n ¼ 5). Table 2. Parameters based on statistical moment theory for CsA in tear fluid after topical administration in conscious rabbits (n ¼ 6). Pharmacokinetics parameters AUC (mg*h/mL) AUMC (mg*h2/mL) MRT (h)
CsA emulgel group
CsA castor oil group
18.21** 155.13** 8.52*
8.31 55.89 6.72
AUC, area under the curve; MRT, mean retention time; AUMC, area under the moment curve. **p50.01, *p50.05.
Formulation and evaluation of CsA emulgel for ocular delivery
DOI: 10.3109/10717544.2013.861883
release where the amount of released drug after 48 h was more than 80%, which is consistent with previously-reported results (Du et al., 2011). As the concentration of polycarbophil increased, the amount of the drug released from the emulgel decreased. All the f2 values obtained between the two formulations (0.2% and 0.25%, 0.25% and 0.3%) were lower than the limit value of 50, indicating dissimilarity between the two dissolution profiles. This finding may be attributed to Table 3. Mean tear concentration of CsA after instillation of E-gel preparation and castor oil preparation in rabbit(mg/ml, X SD, n ¼ 6).
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Time (h) 0.08 0.50 1.00 2.00 3.50 7.00 14.00 24.00
CsA E-gel group (mg/g)
CsA castor oil group (mg/g)
3.97 0.74 3.07 0.64 2.54 0.16 1.89 0.50 1.29 0.24 0.69 0.06 0.29 0.05 0.18 0.02
3.05 0.92 1.78 0.25 2.86 0.96 2.03 0.52 1.40 0.49 0.90 0.30 0.52 0.07 0.47 0.43
5
the different viscosity between the three emulgel formulations. When the viscosity is too high, the diffusion of the entrapped drug from the network structure of polycarbophil might be difficult; furthermore, the emulgel might be undroppable from the package, resulting in low spread on the eye surface. The drug release data were analyzed according to zero- and first-order kinetics as well as diffusion-controlled mechanism using linear regression analysis. The results revealed that the CsA released from the emulgel formulations followed Higuchi’s diffusion model with a correlation coefficient ranging from 0.9876 to 0.9932, which means an excellent model fit. This finding indicates that the rate-controlling stage in the release process was diffusion of the dissolved drug through the gel network to the external medium, which, in turn, interprets why the higher amount of the polycarbophil cause the lower drug release (Balasubramaniam et al., 2003). The in vitro release study conditions might have been different from those possibly met by the emulgels when they were instilled into eyes. However, the results showed that the
Figure 4. Light microscopy of rabbit’s iris (A, B), rabbit’s conjunctiva (C, D), and rabbit’s corneal tissue sections (E, F), from CsA emulgel-treated (OD) and control (OS) rabbit eyes. Samples and tissue sections from treated eyes showed no alterations in morphological details compared to control eyes. Eye tissues from CsA emulgel-treated animals gave similar results. Staining: (A–F) hematoxylin-eosin. Scale bar ¼ 100 mm.
6
Y. Shen et al.
formed emulgels had the ability to retain CsA for the duration of the study (48 h). In the cul-de-sac, the emulgels would probably undergo rapid dissolution due to the shearing action of the eyelid and eyeball movements.
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Precorneal clearance study The precorneal CsA concentration-time profiles of emulgel and castor oil preparations in conscious rabbits are depicted in Figure 3, where a burst release was observed in castor oil group but the clearance of CsA in the emulgel group was much slower. Table 2 summarizes the results of the noncompartmental analysis. The area under the curve (AUC) of CsA emulgel preparation was significantly higher than that of castor oil preparation (p50.01). And the mean residence time (MRT) of CsA emulgel preparation was significantly higher than that of castor oil preparation (p50.05). The Tmax and Cmax are shown in Table 3. These results suggested that emulgel preparations containing the viscous materials cause the drug to be eliminated slowly from the precorneal area, which could increase the precorneal residence and thus improve the ocular bioavailability. In vivo tolerance assay After 168 h of the first instillation of the CsA emulgels, conjunctiva, iris and cornea samples were taken from both eyes. No significant differences were observed between control and treated eyes for each group (Figure 4). Pathology of conjunctival, iridal and corneal epithelia displayed normal numbers of cell layers with cells maintaining normal morphology. Goblet cells remained abundant and filled with the secretory product. Conjunctiva-associated lymphoid tissue was identified in all conjunctivas and no differences in size or location were observed in CsA emulgels-exposed eyes compared to control eyes (Figure 4). In addition, scattered polymorphonuclear cells were identified in the conjunctival stroma, and no signs of tissue edema were observed in the cornea, conjunctiva or iris. Hence, given that microscopic observations of the eyes treated with CsA emulgels did not reveal any lesions, the CsA emulgels could be considered as rarely causing irritation to the eyes of the rabbits.
Conclusion In the coming years, topical drug delivery is expected to be extensively used for better patient compliance and tolerance. Since emulgel is helpful in enhancing spreadability, adhesion, viscosity and extrusion, this novel drug delivery system garners more and more attention. In addition, emulgel is also favorable to become a solution for loading hydrophobic drugs in water-soluble gel bases for the long-term stability. Moreover, to the best of our knowledge, no investigation on CsA emulgel formulations have been reported before. Based on the above results, which showed that CsA emulgel formulations prepared with polycarbophil exhibited acceptable physical properties and drug release, remaining unchanged upon storage for 3 months, we can arrive at the conclusion that polycarbophil-based emulgel is able to extend the retention time on the ocular surface as well as improve the
Drug Deliv, Early Online: 1–7
ocular bioavailability without any lesions. Therefore, the polycarbophil-based emulgel is proved to be an ideal choice for topical drug delivery in ophthalmic therapy.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by National Natural Science Foundation of China (No. 81201182, 81072588) and Fundamental Research Funds for the Central Universities (Program No. ZJ13056).
References Balasubramaniam J, Kant S, Pandit JK. (2003). In vitro and in vivo evaluation of the Gelrite gellan gum-based ocular delivery system for indomethacin. Acta Pharm 53:251–61. Belin MW, Bouchard CS, Phillips TM. (1990). Update on topical cyclosporin A. Background, immunology, and pharmacology. Cornea 9:184–95. Benitez del Castillo JM, del Aguila C, Duran S, et al. (1994). Influence of topically applied cyclosporine A in olive oil on corneal epithelium permeability. Cornea 13:136–40. Bother H, Waaler T. (1990). Rheological characterization of tear substitutes. Drug Dev Ind Pharm 16:755–68. Chen Y, Lu Y, Zhong Y, et al. (2012). Ocular delivery of cyclosporine A based on glyceryl monooleate/poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation. J Drug Target 20:856–63. Costa P. (2001). An alternative method to the evaluation of similarity factor in dissolution testing. Int J Pharm 220:77–83. Diebold Y, Jarrin M, Saez V, et al. (2007). Ocular drug delivery by liposome-chitosan nanoparticle complexes (LCS-NP). Biomaterials 28:1553–64. Du S, Sun CM, Shen Y, et al. (2011). Study on ophthalmic emulsion-gel of Cyclosporine A. Pharm Clin Res 19:215–19. Er Y, Barnes TJ, Fornasiero D, et al. (2009). The encapsulation and release of guanosine from PEGylated liposomes. J Liposome Res 19: 29–36. Gupta V, Bansal P, Gupta A, et al. (2010). Formulation and evaluation of topical gel of diclofenac sodium using different polymers. Drug Invention Today 2:250–3. Kishore AS, Surekha P, Murthy PB. (2009). Assessment of the dermal and ocular irritation potential of multi-walled carbon nanotubes by using in vitro and in vivo methods. Toxicol Lett 191:268–74. Krenzer KL, Zhang JZ, Coffey MJ, et al. (2012). Safety of repeated topical ocular administration of a polycarbophil-based formulation in several models of ocular surgery in rabbits. J Cataract Refract Surg 38: 696–704. Kumar L, Verma R. (2011). In vitro evaluation of topical gel prepared using natural polymer. Int J Drug Deliv 2:58–63. Lallemand F, Felt-Baeyens O, Besseghir K, et al. (2003). Cyclosporine A delivery to the eye: a pharmaceutical challenge. Eur J Pharm Biopharm 56:307–18. Marquardt D, Sucker H. (1998). Oil-in water-emulsion gels: determination and mathematical treatment of flow properties. Eur J Pharm Biopharm 46:115–24. Mohamed MI. (2004). Optimization of chlorphenesin emulgel formulation. AAPS Journal 6:e26–32. Mondon K, Zeisser-Laboue`be M, Gurny R, et al. (2011). Novel Cyclosporin A formulations using MPEG–hexyl-substituted polylactide micelles: a suitability study. Eur J Pharm Biopharm 77:56–65. Niazi SK. (2009). Stability testing of new drug substances and products. In: Niazi SK, ed. Handbook of pharmaceutical manufacturing formulations. (second ed.). New York: Informa Healthcare, 52–9. Parrott EL. (1970). Pharmaceutical technology: fundamental pharmaceutics. Lincoln, USA: Burgess Publishing Company. Perioli L, Pagano C, Mazzitelli S, et al. (2008). Rheological and functional characterization of new antiinflammatory delivery systems designed for buccal administration. Int J Pharm 356:19–28.
DOI: 10.3109/10717544.2013.861883
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Rodriguez-Aller M, Kaufmann B, Guillarme D, et al. (2012). In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease. Eur J Pharm Biopharm 80:544–52. Schreiber SL, Crabtree GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunol Today 13:136–42. Schumacher A, Nordheim A. (1992). Progress towards a molecular understanding of cyclosporin A-mediated immunosuppression. Clin Investig 70:773–9. Shahin M, Hady SA, Hammad M, et al. (2011). Novel jojoba oil-based emulsion gel formulations for clotrimazole delivery. APS PharmSciTech 12:239–47. Steiger M. (2010). Topical emulsion-gel composition comprising diclofenac sodium. US Patent 7,732,489 B2.
Formulation and evaluation of CsA emulgel for ocular delivery
7
Stevenson D, Tauber J, Reis BL. (2000). Efficacy and safety of cyclosporin A ophthalmic emulsion in the treatment of moderate-to-severe dry eye disease: a dose-ranging, randomized trial. The Cyclosporin A Phase 2 Study Group. Ophthalmology 107:967–74. Van Haeringen NJ. (1981). Clinical biochemistry of tears. Surv Ophthalmol 26:84–96. Van Ooteghem MMM. (1993). Formulation of ophthalmic solutions and suspensions. Problems and advantages. In: Edman P, ed. Biopharmaceutics of ocular drug delivery. Boca Raton, FL: CRC Press, 27–42. Wei G, Xu H, Ding PT, et al. (2002). Thermosetting gels with modulated gelation temperature for ophthalmic use: the rheological and gamma scintigraphic studies. J Control Release 83:65–74.
Formulation and evaluation of Cyclosporin A emulgel for ocular delivery Yan Shen1, Xiang Ling1, Weiwei Jiang2, Shuang Du1, Yang Lu3, and Jiasheng Tu1 Department of Pharmaceutics, China Pharmaceutical University, Nanjing, China, 2Patent Examination Cooperation Center of SIPO, Jiangsu, Suzhou, China, and 3Department of Industrial Pharmacy, School of Chinese Pharmacy, Beijing University of TCM, Beijing, China
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
1
Abstract
Keywords
Emulgels have been extensively covered as a promising drug delivery system for the administration of lipophilic drugs. This work was conducted to develop an emulgel formulation for Cyclosporin A (CsA) employing polycarbophil as the gelling agent for ocular delivery. The prepared emulgels were evaluated for their physical appearance, rheological behavior, drug release, stability, precorneal clearance and irritation. Results showed that CsA emulgel formulations prepared with polycarbophil exhibited acceptable physical properties and drug release, which remained consistent after storage for 3 months. A prolonged retention time was also observed on the ocular surface with improved ocular bioavailability and no irritation. Therefore, the polycarbophil-based emulgel could be exploited as a potential hydrophobic drug carrier for topical ocular drug delivery.
Cyclosporin A, emulgel, ocular delivery, polycarbophil, precorneal clearance
Introduction A gel is a jelly-like material with a three-dimensional crosslinked network of colloidal solid particles within which miraculous amounts of aqueous or hydro alcoholic liquid could be entrapped (Kumar & Verma, 2011). It is superior in terms of the application as a dosage form and patient acceptability. Despite many advantages, a major obstacle for gels is in the delivery of hydrophobic drugs. So to conquer this impediment, emulgels have emerged as one of the most interesting controlled release drug delivery systems which possess the properties of both emulsions and gels (Steiger, 2010). In the past decade, scientists and industrial researchers have taken increasing interests in the field of pharmaceutical semisolid dosage forms especially emulgels, primarily due to their homogenous behavior and jelly-like consistency. Emulgels, also known as o/w emulsion gels or cream gels, are colloidal systems mixed with oil-in-water solutions as an interior phase. Despite their utilization in cosmetics for a long period, wide application of such hydrophilic systems in pharmaceutics starts with dermatological formulations (Marquardt & Sucker, 1998). In recent years, discovery of novel polymers with diverse functions of improved gelling capacity and as emulsifiers and thickeners dramatically facilitated the development of emulgels; which decreased superficial and interfacial tension, and increased the viscosity
Address for correspondence: Yan Shen, Department of Pharmaceutics, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China. Tel: 0086-25-83271305. Fax: 0086-25-83271351. Email: shenyan [email protected] Yang Lu, Department of Industrial Pharmacy, School of Chinese Pharmacy, Beijing University of TCM, 6 Zhong Huan Nan Lu, Beijing 100102, China. Email: [email protected]
History Received 27 August 2013 Revised 30 October 2013 Accepted 30 October 2013
of the aqueous phase, resulting in stable emulsions and creams (Gupta et al., 2010). Eventually, their rheological properties can be controlled easily by the proper selection of the gelling agent as well as the oily ingredient. CsA, which has a selective immunosuppressive effect, is used for several autoimmune as well as contingent eye diseases for its specific inhibition on the T-cell-dependent immune reactions at high-rate (Belin et al., 1990; Schreiber & Crabtree, 1992; Schumacher & Nordheim, 1992). CsA is a pretty hydrophobic drug with poor water solubility (0.012 mg/mL at 25 C) (Mondon et al., 2011), hence, creating formulation problems. To yield an aqueous CsA formulation, many approaches have been attempted such as co-solvent, pro-drugs, preparation in different oils (e.g. castor, olive or peanut) or emulsions (Stevenson et al., 2000; Chen et al., 2012; Rodriguez-Aller et al., 2012). Nevertheless, the use of these approaches is generally limited by the appearance of side-effects which may include itching, redness, burning and toxic effects on the cornea (Benitez del Castillo et al., 1994; Lallemand et al., 2003). An eye drop solution is the best choice for topical therapy of ocular diseases, notably, in some cases, when a localized action (e.g., the cornea and/or anterior chamber) of drug is needed. However, self-protecting mechanisms, such as lacrimal secretion and blinking reflex, along with corneal impermeability, make drug retention time so short that frequent administration is necessary for highly efficient therapy (Wei et al., 2002). In order to avoid the rapid dilution, formulations with an increased viscosity have been investigated. The increase of both the residence time of drug in the precorneal area and the absorption can improve its therapeutic effect. Recently, Durasite (Insite Vision, Inc.), a polycarbophilbased optical formulation (polycarbophil 0.9%) with
2
Y. Shen et al.
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
azithromycin 1.0% (Azasite) and besifloxacin 0.6% (Besivance) for the treatment of bacterial conjunctivitis, was introduced into the market. In addition, after a long-term topical ophthalmic administration on rabbits, the polycarbophil-based formulation was proved to be harmless for chronic use as well as impressive in improving the residence time of drug on the ocular surface, thus a promising ophthalmic medicinal preparation for the treatment of eye diseases (Krenzer et al., 2012). Based on these considerations and our preliminary studies, the aim of this work was to develop an emulgel formulation of CsA using polycarbophil as gelling agent. The physical, rheological characteristic, stability under several conditions, in vitro release, eye irritation studies, and the residence time of the drug in the precorneal area of the prepared emulgels were all evaluated.
Materials and methods Materials and animals Cyclosporine A (CsA) was purchased from Galena (Czech Republic). Polycarbophil was obtained from The Lubrizol Corporation (Ohio Clif Lancaster, USA). Poloxamer188 was supplied by Nanjing Well Chemical Co., Ltd (Nanjing, China). Castor oil (analytical reagent) was purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Dialysis membrane was purchased from Sigma Chemical Company (Shanghai, China). Methanol and acetonitrile were of HPLC grade and other chemicals were all of analytical reagent grade. Rabbits were purchased from the Experimental Animal Center of Nanjing Qinglongshan (Nanjing, China). The animals were acclimatized at 25 1 C and 70 5% relative humidity under natural light/dark conditions for 1 week before experiments. All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of China Pharmaceutical University.
Drug Deliv, Early Online: 1–7
to the sample was performed four times using a Sonicator B12 (Branson Sonic Power Company, Danbury, CT). After each sonication, the dispersion was mixed manually. At last, the emulgel system was jellified by adding different volume ratios of the CsA O/W emulsion and the polycarbophil dispersion was then sheared at 200 rpm for 45 min. The pH of the emulgel was regularly adjusted to 6.8 using NaOH or HCl (Mohamed, 2004; Shahin et al., 2011) to maintain its viscosity. The product of emulgel was formulated by now. The formulations were repeated for three times. Evaluation of emulgel Physical examination. The prepared emulgel formulations
were examined for their physical characteristics including color, homogeneity and phase separation. pH measurements: pH was measured at room temperature using a Scientific Instruments model IQ150 pH meter with a PH17-SS pH electrode (Scientific Instruments, Inc., San Diego, California, USA). Osmolarity measurements: Freezing point depression method (Micro-Osmometer Model 3300, Advanced Instruments, Norwood, MA) was used to obtain osmotic pressure (P) versus osmolality curve for CsA. All measurements were done in duplicate.
Methods
Viscosity determination of the prepared formulations was carried out on a cone (0.8 ) and plate geometry viscometer (Brookfield, USA) using spindle SC4-16. Viscosity of sample solutions was measured at different angular velocities at 37 1 C. A typical run comprised changing the angular velocity from 25 to 220 rpm at a controlled ramp speed. After 6 s at 25 rpm, the velocity was increased to 220 rpm with a similar wait time at each speed. The hierarchy of angular velocity was reversed (from 220 to 25 rpm) with a similar wait time of 6 s. Eventually, the average of two readings was used to calculate the viscosity. Evaluations were conducted in triplicate (Perioli et al., 2008).
Preparation of emulgels
Stability studies
The CsA cannot be easily dissolved in castor oil, in order to obtain the O/W emulsion, 100 mg CsA was added to 1 ml ethanol and 1.5 g castor oil was then added to form a homogeneous oil phase. After evaporation of ethanol under vacuum with a rotary evaporator, the oil phase of CsA dissolved in castor oil was obtained. Afterwards, the aqueous phase was prepared by dissolving 330 mg poloxamer 188 in ultrapure water. In the next step, both oil and aqueous phase were mixed together under continuous magnetic stirring at room temperature. Finally, the O/W emulsion was formed with a homogenizer (Erweka, type AR 401, Heusenstamm, Germany) for 5 min at 10 000 rpm. For the purpose of preparing the polycarbophil dispersion, 0.2 g of the polymer powder was dispersed in an iso-osmotic solution containing 3.0% (w/v) glycerol. pH was adjusted to physiological pH 6.8 by adding the required amount of NaOH. To ensure complete hydration, the solutions were stored at 6 C for a minimum of 12 h. And then, an additional sonication procedure of 30 s by imposing 80 W power output
The prepared CsA emulgel formulations were stored away from light in high-density polyethylene bottles at 40 C and 4 C for 3 months, respectively. Samples were withdrawn at 15-day time intervals and evaluated for their physical appearance, pH, osmotic pressure, rheological behavior and drug content (Niazi, 2009).
Rheological study.
Drug release study in simulated lachrymal fluid Simulated lachrymal fluid (SLF) is an electrolyte solution containing 1.7893 g/L KCl, 6.3118 g/L NaCl, 2.1842 g/L NaHCO3, 44.4 mg/L CaCl2 and 47.6 mg/L MgCl2 (Van Haeringen, 1981). Physiological pH (7.4 0.1) was adjusted by adding the required amount of 0.1 mol/L HCl. Due to the poor solubility of CsA in the water, the SLF containing 30% ethanol was used as the release medium. CsA emulgel release kinetics were described by using a dialysis-bag method (Er et al., 2009). In brief, the CsA emulgel (1 g) was added to the dialysis bag (cut-off
Formulation and evaluation of CsA emulgel for ocular delivery
DOI: 10.3109/10717544.2013.861883
weight: 3500 Da) and then placed at the bottom of the dissolution meter (100 rpm) at 34 C. The mount of the drug adsorbed by the dialysis bag was negligible. Each dissolution cup was added with 30 mL release medium. After that, the amount of CsA released from the emulgel was determined by HPLC at predetermined time points. 2.0 mL sample was withdrawn from the dissolution cup, and an equal volume of the release medium was refilled to maintain the original volume of the dissolution cup. Each experiment was run six times. CsA release was expressed as below (Equation 1) and plotted as a function of time: CsA release % ¼
Amount of CsA released to the medium Total amount of CsA in the dialysis bag 100%
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
ð1Þ Baseline studies on free CsA solutions have shown that free drug transport through the dialysis membrane is fast, in comparison to that released from emulgel while the nonspecific free CsA adsorption on the dialysis membrane was negligible. The in vitro release profiles of CsA emulgels were compared using similarity factors, f2, as defined by the following Equation (2) (Costa, 2001): ( ) 0:5 1 Xn 2 f2 ¼ 50 log 1 þ ðR Tt Þ 100 ð2Þ t1 t n where n is number of time points, and Tt and Rt are percentage releases at time point (t) for the reference and test formulation, respectively. Cyclosporine A quantification CsA concentrations were determined by the HPLC method. The HPLC system employed in the study consisted of an Agilent G1100 HPLC series system (Agilent Technologies, Palo Alto, CA) equipped with an Agilent G1310A pump and an Agilent G1314A variable wavelength UV-VIS photo diode array detector (PDAD) set at 210 nm. A Waters Xbridge-C18 ODS column (150 mm 4.6 mm, 5 mm) was operated at 70 C due to the existence of various conformers of CsA. The mobile phase consisted of acetonitrile–water–methanol–phosphoric acid (650:300:50:0.4, v/v/v/v) was flowing at a rate of 0.6 mL/min. A calibration curve was established on each running day. These curves showed good linearity, with correlation coefficients no less than 0.99. The limit of quantification (LOQ) and detection (LOD) were 50 and 20 ng/mL, respectively. Precorneal clearance study Ten rabbits divided into two groups (five rabbits in each group) were used to determine precorneal clearance. For emulgel group, each rabbit was instilled with 50 mL CsA emulgel preparation according to the section ‘‘Preparation of emulgels’’ (0.05%) on the right eye, and 50 mL blank saline solution on the left eye, respectively. For castor oil group, each rabbit was instilled with 50 mL CsA castor oil ophthalmic solution (0.05%) on the right eye, and 50 mL blank saline solution on the left eye, respectively. At 0, 0.5, 1, 2, 4, 7, 14,
3
24 h after instillation, tear samples of both eyes were collected by Schirmer test strips. The amount of tear withdrawn was calculated by subtracting the weight of each strip after sampling from the weight before sampling. Subsequently, the Schirmer strip was placed into an Eppendorf tube, dried under N2 stream, and then 0.2 mL of mobile phase was added. The sample was vortexed for 30 s thoroughly to dissolve CsA into the mobile phase and centrifuged under 300 g. All the experiment were performed under the Protocol of Ethical Comittee Permission. Pharmacokinetic and statistical analysis Pharmacokinetic analysis was accomplished using the 3p87 Pharmacokinetic Program (Chinese Society of Mathematical Pharmacology 1987, China). Statistical comparisons were performed using analysis of variance (ANOVA) or the Student’s t-test, where appropriate, and statistical significance was set at p50.05. In vivo eye irritation assessment in rabbits None of the rabbits exhibited ocular defects or pre-existing corneal injury in the pre-experimental inspection using an ophthalmoscope 24 h prior to administration. After rabbits were randomly divided into two groups with five in each, approximately 50 mL of CsA emulgel (0.05%) and 0.9% saline solution was dropped into the conjunctival sac of the left eye of each animal. The lids were then gently held together for about 1 s in order to prevent loss of the material. The right eye, which remained untreated, served as control. Animals wore Elizabethan collars during the study to avoid licking and scratching their eyes and faces. The rabbits’ eyes were instilled on day 1, 2, 3, 4 and 7. Then residues of the prepared formulations were removed from eyes by washing with 0.9% saline solution at 168 h (7 days) post-administration (Kishore et al., 2009). The animals were then euthanized by air embolism after being deeply anesthetized with an intramuscular overdose of anesthetic and paralyzing mixture of xylazine (20 mg/kg) and ketamine (200 mg/kg). Eyeball and lid tissues were removed and fixed in Davison’s fixative solution composed of 1 part glacial acetic acid, 2 parts 37% formaldehyde, 3 parts 95% ethanol, and 3 parts distilled water. Following fixation, they were embedded in paraffin for pathology. Eyeballs were determined in a masked fashion on the basis of following criteria: variation of the ocular surface epithelial cells, edema in lid tissues, presence of inflammatory cells, and any other abnormality (Diebold et al., 2007).
Results and discussion Physical appearance The prepared CsA emulgel formulations were white viscous creamy preparations with smooth and homogeneous appearance. They were easily droppable with acceptable viscosity and fair mechanical properties. The pH value of all the prepared formulations ranged from 6.3 to 6.5 and the osmotic pressure values ranged from 245 to 310 Osmol/kg, which are considered acceptable to avoid the risk of irritation upon application to the eye.
4
Y. Shen et al.
Drug Deliv, Early Online: 1–7
Table 1. The stability of CsA emulgel. Month
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Content Viscosity (cP) pH Osmotic pressure (mOsmol/kg) Appearance
0
1
3
99.85% 1888 6.66 301
99.57% 1917 6.67 308
99.63% 1924 6.66 303
Milk white, not layed
Milk white, not layed
Milk white, not layed
Figure 1. Viscosity-shear rate profiles of CsA emulgel.
Rheological studies The tests were performed at a rotating speed ranging from 25 to 220 rpm for 20 min. Results are shown in Figure 1. The formulations exhibited pseudo-plastic rheology as evinced by shear thinning and an increase in the shear stress with enhanced angular velocity, which are consistent with our preliminary data obtained during formulation screening (Du et al., 2011). In this flow behavior, static molecules could be entrapped in the cross-linked network. When sheared to decrease the flow resistance, the molecules would be realigned along the direction of flow, thus resulting in the lower viscosity (Parrott, 1970). The viscosity of the formulations prepared was (2312 25) cp with SC16 rotor at 50 rpm. Shear thinning behavior is a desirable property of topical ophthalmic preparations since it should thin during application and thicken otherwise (Van Ooteghem, 1993). Since the ocular shear rate is very high, ranging from 0.03 s1 during inter-blinking periods to 4250–28 500 s1 during blinking (Bother & Waaler, 1990), viscoelastic fluids with a viscosity that is high under the low shear rate conditions and low under the high shear rate conditions are often preferred, which could spread easily on the ocular surface and then increase the ocular bioavailability as well as the compliance of patients.
Figure 2. The effects of concentration and viscosity of gel on the release behavior of CsA emulgel.
Stability study The physical appearance, pH, osmotic pressure, rheological properties and drug content were investigated to evaluate the stability of the prepared formulations, and no significant changes were observed (Table 1). The results showed that all emulgel formulations stored for up to 3 months were as stable as the newly prepared ones. In vitro release study To make a deep comparison, we investigated the in vitro release behaviors of three emulgel formulations with varying polycarbophil contents of 0.2%, 0.25% and 0.3%. As shown in Figure 2, the 0.2% polycarbophil emulgel formulation with the lowest viscosity was observed to exhibit the best drug
Figure 3. Concentration–time profiles of CsA in tear fluid after instillation in conscious rabbits (n ¼ 5). Table 2. Parameters based on statistical moment theory for CsA in tear fluid after topical administration in conscious rabbits (n ¼ 6). Pharmacokinetics parameters AUC (mg*h/mL) AUMC (mg*h2/mL) MRT (h)
CsA emulgel group
CsA castor oil group
18.21** 155.13** 8.52*
8.31 55.89 6.72
AUC, area under the curve; MRT, mean retention time; AUMC, area under the moment curve. **p50.01, *p50.05.
Formulation and evaluation of CsA emulgel for ocular delivery
DOI: 10.3109/10717544.2013.861883
release where the amount of released drug after 48 h was more than 80%, which is consistent with previously-reported results (Du et al., 2011). As the concentration of polycarbophil increased, the amount of the drug released from the emulgel decreased. All the f2 values obtained between the two formulations (0.2% and 0.25%, 0.25% and 0.3%) were lower than the limit value of 50, indicating dissimilarity between the two dissolution profiles. This finding may be attributed to Table 3. Mean tear concentration of CsA after instillation of E-gel preparation and castor oil preparation in rabbit(mg/ml, X SD, n ¼ 6).
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Time (h) 0.08 0.50 1.00 2.00 3.50 7.00 14.00 24.00
CsA E-gel group (mg/g)
CsA castor oil group (mg/g)
3.97 0.74 3.07 0.64 2.54 0.16 1.89 0.50 1.29 0.24 0.69 0.06 0.29 0.05 0.18 0.02
3.05 0.92 1.78 0.25 2.86 0.96 2.03 0.52 1.40 0.49 0.90 0.30 0.52 0.07 0.47 0.43
5
the different viscosity between the three emulgel formulations. When the viscosity is too high, the diffusion of the entrapped drug from the network structure of polycarbophil might be difficult; furthermore, the emulgel might be undroppable from the package, resulting in low spread on the eye surface. The drug release data were analyzed according to zero- and first-order kinetics as well as diffusion-controlled mechanism using linear regression analysis. The results revealed that the CsA released from the emulgel formulations followed Higuchi’s diffusion model with a correlation coefficient ranging from 0.9876 to 0.9932, which means an excellent model fit. This finding indicates that the rate-controlling stage in the release process was diffusion of the dissolved drug through the gel network to the external medium, which, in turn, interprets why the higher amount of the polycarbophil cause the lower drug release (Balasubramaniam et al., 2003). The in vitro release study conditions might have been different from those possibly met by the emulgels when they were instilled into eyes. However, the results showed that the
Figure 4. Light microscopy of rabbit’s iris (A, B), rabbit’s conjunctiva (C, D), and rabbit’s corneal tissue sections (E, F), from CsA emulgel-treated (OD) and control (OS) rabbit eyes. Samples and tissue sections from treated eyes showed no alterations in morphological details compared to control eyes. Eye tissues from CsA emulgel-treated animals gave similar results. Staining: (A–F) hematoxylin-eosin. Scale bar ¼ 100 mm.
6
Y. Shen et al.
formed emulgels had the ability to retain CsA for the duration of the study (48 h). In the cul-de-sac, the emulgels would probably undergo rapid dissolution due to the shearing action of the eyelid and eyeball movements.
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Precorneal clearance study The precorneal CsA concentration-time profiles of emulgel and castor oil preparations in conscious rabbits are depicted in Figure 3, where a burst release was observed in castor oil group but the clearance of CsA in the emulgel group was much slower. Table 2 summarizes the results of the noncompartmental analysis. The area under the curve (AUC) of CsA emulgel preparation was significantly higher than that of castor oil preparation (p50.01). And the mean residence time (MRT) of CsA emulgel preparation was significantly higher than that of castor oil preparation (p50.05). The Tmax and Cmax are shown in Table 3. These results suggested that emulgel preparations containing the viscous materials cause the drug to be eliminated slowly from the precorneal area, which could increase the precorneal residence and thus improve the ocular bioavailability. In vivo tolerance assay After 168 h of the first instillation of the CsA emulgels, conjunctiva, iris and cornea samples were taken from both eyes. No significant differences were observed between control and treated eyes for each group (Figure 4). Pathology of conjunctival, iridal and corneal epithelia displayed normal numbers of cell layers with cells maintaining normal morphology. Goblet cells remained abundant and filled with the secretory product. Conjunctiva-associated lymphoid tissue was identified in all conjunctivas and no differences in size or location were observed in CsA emulgels-exposed eyes compared to control eyes (Figure 4). In addition, scattered polymorphonuclear cells were identified in the conjunctival stroma, and no signs of tissue edema were observed in the cornea, conjunctiva or iris. Hence, given that microscopic observations of the eyes treated with CsA emulgels did not reveal any lesions, the CsA emulgels could be considered as rarely causing irritation to the eyes of the rabbits.
Conclusion In the coming years, topical drug delivery is expected to be extensively used for better patient compliance and tolerance. Since emulgel is helpful in enhancing spreadability, adhesion, viscosity and extrusion, this novel drug delivery system garners more and more attention. In addition, emulgel is also favorable to become a solution for loading hydrophobic drugs in water-soluble gel bases for the long-term stability. Moreover, to the best of our knowledge, no investigation on CsA emulgel formulations have been reported before. Based on the above results, which showed that CsA emulgel formulations prepared with polycarbophil exhibited acceptable physical properties and drug release, remaining unchanged upon storage for 3 months, we can arrive at the conclusion that polycarbophil-based emulgel is able to extend the retention time on the ocular surface as well as improve the
Drug Deliv, Early Online: 1–7
ocular bioavailability without any lesions. Therefore, the polycarbophil-based emulgel is proved to be an ideal choice for topical drug delivery in ophthalmic therapy.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by National Natural Science Foundation of China (No. 81201182, 81072588) and Fundamental Research Funds for the Central Universities (Program No. ZJ13056).
References Balasubramaniam J, Kant S, Pandit JK. (2003). In vitro and in vivo evaluation of the Gelrite gellan gum-based ocular delivery system for indomethacin. Acta Pharm 53:251–61. Belin MW, Bouchard CS, Phillips TM. (1990). Update on topical cyclosporin A. Background, immunology, and pharmacology. Cornea 9:184–95. Benitez del Castillo JM, del Aguila C, Duran S, et al. (1994). Influence of topically applied cyclosporine A in olive oil on corneal epithelium permeability. Cornea 13:136–40. Bother H, Waaler T. (1990). Rheological characterization of tear substitutes. Drug Dev Ind Pharm 16:755–68. Chen Y, Lu Y, Zhong Y, et al. (2012). Ocular delivery of cyclosporine A based on glyceryl monooleate/poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation. J Drug Target 20:856–63. Costa P. (2001). An alternative method to the evaluation of similarity factor in dissolution testing. Int J Pharm 220:77–83. Diebold Y, Jarrin M, Saez V, et al. (2007). Ocular drug delivery by liposome-chitosan nanoparticle complexes (LCS-NP). Biomaterials 28:1553–64. Du S, Sun CM, Shen Y, et al. (2011). Study on ophthalmic emulsion-gel of Cyclosporine A. Pharm Clin Res 19:215–19. Er Y, Barnes TJ, Fornasiero D, et al. (2009). The encapsulation and release of guanosine from PEGylated liposomes. J Liposome Res 19: 29–36. Gupta V, Bansal P, Gupta A, et al. (2010). Formulation and evaluation of topical gel of diclofenac sodium using different polymers. Drug Invention Today 2:250–3. Kishore AS, Surekha P, Murthy PB. (2009). Assessment of the dermal and ocular irritation potential of multi-walled carbon nanotubes by using in vitro and in vivo methods. Toxicol Lett 191:268–74. Krenzer KL, Zhang JZ, Coffey MJ, et al. (2012). Safety of repeated topical ocular administration of a polycarbophil-based formulation in several models of ocular surgery in rabbits. J Cataract Refract Surg 38: 696–704. Kumar L, Verma R. (2011). In vitro evaluation of topical gel prepared using natural polymer. Int J Drug Deliv 2:58–63. Lallemand F, Felt-Baeyens O, Besseghir K, et al. (2003). Cyclosporine A delivery to the eye: a pharmaceutical challenge. Eur J Pharm Biopharm 56:307–18. Marquardt D, Sucker H. (1998). Oil-in water-emulsion gels: determination and mathematical treatment of flow properties. Eur J Pharm Biopharm 46:115–24. Mohamed MI. (2004). Optimization of chlorphenesin emulgel formulation. AAPS Journal 6:e26–32. Mondon K, Zeisser-Laboue`be M, Gurny R, et al. (2011). Novel Cyclosporin A formulations using MPEG–hexyl-substituted polylactide micelles: a suitability study. Eur J Pharm Biopharm 77:56–65. Niazi SK. (2009). Stability testing of new drug substances and products. In: Niazi SK, ed. Handbook of pharmaceutical manufacturing formulations. (second ed.). New York: Informa Healthcare, 52–9. Parrott EL. (1970). Pharmaceutical technology: fundamental pharmaceutics. Lincoln, USA: Burgess Publishing Company. Perioli L, Pagano C, Mazzitelli S, et al. (2008). Rheological and functional characterization of new antiinflammatory delivery systems designed for buccal administration. Int J Pharm 356:19–28.
DOI: 10.3109/10717544.2013.861883
Drug Delivery Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.
Rodriguez-Aller M, Kaufmann B, Guillarme D, et al. (2012). In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease. Eur J Pharm Biopharm 80:544–52. Schreiber SL, Crabtree GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunol Today 13:136–42. Schumacher A, Nordheim A. (1992). Progress towards a molecular understanding of cyclosporin A-mediated immunosuppression. Clin Investig 70:773–9. Shahin M, Hady SA, Hammad M, et al. (2011). Novel jojoba oil-based emulsion gel formulations for clotrimazole delivery. APS PharmSciTech 12:239–47. Steiger M. (2010). Topical emulsion-gel composition comprising diclofenac sodium. US Patent 7,732,489 B2.
Formulation and evaluation of CsA emulgel for ocular delivery
7
Stevenson D, Tauber J, Reis BL. (2000). Efficacy and safety of cyclosporin A ophthalmic emulsion in the treatment of moderate-to-severe dry eye disease: a dose-ranging, randomized trial. The Cyclosporin A Phase 2 Study Group. Ophthalmology 107:967–74. Van Haeringen NJ. (1981). Clinical biochemistry of tears. Surv Ophthalmol 26:84–96. Van Ooteghem MMM. (1993). Formulation of ophthalmic solutions and suspensions. Problems and advantages. In: Edman P, ed. Biopharmaceutics of ocular drug delivery. Boca Raton, FL: CRC Press, 27–42. Wei G, Xu H, Ding PT, et al. (2002). Thermosetting gels with modulated gelation temperature for ophthalmic use: the rheological and gamma scintigraphic studies. J Control Release 83:65–74.
