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Bimatoprost loaded microemulsion laden contact lens to treat glaucoma
Journal Pre-proof Bimatoprost loaded microemulsion laden contact lens to treat glaucoma: In vitro and in vivo studies Wenwen Xu, Wanzhen Jiao, Shangbin Li, Xiangchen Tao, Guoying Mu PII:
S1773-2247(19)31229-8
DOI:
https://doi.org/10.1016/j.jddst.2019.101330
Reference:
JDDST 101330
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 19 August 2019 Revised Date:
14 October 2019
Accepted Date: 15 October 2019
Please cite this article as: W. Xu, W. Jiao, S. Li, X. Tao, G. Mu, Bimatoprost loaded microemulsion laden contact lens to treat glaucoma: In vitro and in vivo studies, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.101330. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
Bimatoprost loaded microemulsion laden contact lens to treat glaucoma: In vitro and in vivo studies Wenwen Xu a, Wanzhen Jiao a, Shangbin Li b, Xiangchen Tao a, Guoying Mu * a a
Department of Ophthalmology, Shandong Provincial Hospital, Shandong University,
Shandong 250021, China b
Department of Healthcare, Shandong Provincial Hospital, Shandong University,
Shandong 250021,China
* Corresponding author at: Department of Ophthalmology, Shandong Provincial Hospital, Shandong University, Jingwu Road, Huaiyin District, Jinan, Shandong 250021, China. Tel.: +861 8732200909 E-mail address: [email protected] (G. Mu)
Abstract Bimatoprost is widely used to manage glaucoma. Currently, it is delivered via eye drop solution in high doses due to poor ocular bioavailability. The high and daily ocular dosing lead to ocular hyperaemia causing patient non-compliance. The contact lenses can be used to sustain the release of drug by conventional soaking technology, without altering the critical contact lens properties. However, the soaking method showed low drug uptake and high burst release, due to the absence of efficient controlling membrane. Here we investigate the effect of microemulsion on bimatoprost uptake from the soaking solution and its effect on drug release kinetics. The contact lenses were soaked in bimatoprost-microemulsion soaking solution (ME) and compared with the bimatoprost-soaking solution (SM) without microemulsion. The uptake of bimatoprostmicroemulsion in the contact lenses did not altered the swelling, transmittance and folding endurance properties. The two fold increase in the uptake/loading of bimatoprost was noted from the bimatoprost-microemulsion (ME) soaking solution in comparison to bimatoprost-soaking solution (SM). The in vitro flux data of ME batches (up to 96h) showed improvement in the release rate profiles in comparison to SM batches (up to 48h). The in vivo studies in the rabbit tear fluid showed low burst release and improvement in the bimatoprost retention time with ME contact lens in comparison to the SM contact lens and eye drop solution. The study demonstrate the potential of microemulsion to improve the uptake of bimatoprost and sustain release kinetics without altering the critical lens properties of the contact lens.
Key words: Bimatoprost, silicone contact lenses, soaking method, microemulsion, animal studies.
1. Introduction Bimatoprost is widely used to manage glaucoma by the eye drop solution in high doses (1 drop ≈ 15 µg, 0.03%w/v Lumigan eye drop), due to short ocular drug residence time and poor ocular bioavailability [1-5]. The daily high ocular dosing lead to ocular hyperaemia causing patient non-compliance [6-9]. The silicon contact lenses can be used to sustain the release of bimatoprost using soaking technique without affecting the critical contact lens properties [10-12]. However, the soaking method showed low drug uptake and high burst release, due to the absence of efficient controlling membrane [1316]. Thus, an innovative approach is desirable to increase the drug uptake along with sustain release capacity of the contact lens. Several development strategies have been reported to sustain the release of ocular drugs from the contact lenses including soaking method [17-19], polymeric nanoparticles [20-22], molecular imprinting [23-25], PLGA films [26, 27], implantation technology [28, 29], use of supercritical fluids [30], etc. The strategies do showed promising application of the contact lenses, however, they failed in the optimal clinical translation due to alteration in the critical contact lens properties. The soaking method is widely applied to load ophthalmic drugs in the contact lenses. C. M. Phan et al., 2016 loaded moxifloxacin in the silicon contact lenses by soaking method and observed high burst release [31]. C. Chung et al., 2005 developed timolol loaded contact lenses by soaking method, which showed improved drug residence time with low dose [32]. J. Hui et al., 2013 design ciprofloxacin and dexamethasone loaded contact lenses by soaking method using cyclodextrin, which showed high burst release [33]. Andreza et al., 2012 loaded acetazolamide using cyclodextrin pendant contact lens, the system shoed 12 h release [34]. J. Xu et al., 2010 loaded puerarin using β-cyclodextrin in hydrogel to treat glaucoma. The contact lens showed alteration in the swelling and tensile strength with high burst release in flux studies [35]. K. Hui et al., 2015 loaded timolol and dorzolamide by soaking method using Vitamin E contact lens. The system showed 48 h reduction in the IOP in Beagle dogs [36]. C-Chung et al., 2007 used microemulsion to load timolol in the contact lens, which showed sustain release for days [37]. The drug loading by the conventional soaking method does not alter the critical lens properties [38]; however, it
pose the issue of low drug loading and high burst release [14], which is not ideal for the sustain drug delivery. The objective of this paper was to address the issues of conventional soaking technique using microemulsion. The study investigate the effect of bimatoprost loading from the soaking solution and its effect on the drug release kinetics from the contact lens. The contact lenses were soaked in bimatoprost-microemulsion soaking solution and compared with the bimatoprost-soaking solution without microemulsion. The bimatoprost loaded contact lenses were evaluated for in vitro and in vivo studies; to investigate the effect of microemulsion on the bimatoprost uptake and its release kinetics from the contact lenses.
2. Materials and Methods 2.1 Materials Hydroxyl ethylmethacrylate (HEMA), ethyleneglycol dimethacrylate (EGDMA), Irgacure®, dimethyl acrylamide (DMA), and Siloxane (TRIS) were purchased from Sigma-Aldrich Chemicals (MO, USA). Bimatoprost was purchased from Guangzhou Quanao Chemical Co., Ltd. (Guangdong, China). All other reagents and chemicals were purchased from Sigma-Aldrich Chemicals (MO, USA).
2.2 Preparation of microemulsion The microemulsion batches were selected from the pseudo ternary phase diagrams [Smix (1:4 and 1:6) ratio] (supplementary material 1) and the composition are shown in table 1. The Smix solution (Pluronic F68 and Tween 20) was dissolved in required volume of water with constant stirring at room temperature. The required quantity of isopropyl myristate was added drop by drop at 50 RPM to get clear transparent microemulsion [39-41]. One batch of microemulsion was selected to load bimatoprost for further studies based on thermodynamic stability results (section 3.1). Bimatoprost was dissolved in the selected microemulsion at three different levels (0.5 mg/ml, 1 mg/ml and 1.5 mg/ml) to use/incorporate in the soaking solution to load bimatoprost in the contact lens (see section 2.5).
Table 1 Composition and coding of microemulsion. Water (%) Codes Isopropyl myristate (%) Smix solution (%) ME-1(1:4) 1.5 50 48.5 ME-2(1:4) 3 50 47 ME-3(1:6) 1.5 50 48.5 ME-4(1:6) 3 50 47 Smix = Pluronic F68: Tween 20, ME = microemulsion, 1:4 and 1:6 are Smix ratios 2.3. Characterization of microemulsions 2.3.1. Globule size and zeta potential The globule size and zeta potential of microemulsions were determined by Malvern Zetasizer (3000HSA, UK) at 37oC. Sample microemulsion was placed in standard glass cuvette and size was determined at 90o incidence angle [42, 43]. Reading was recorded in triplicate.
2.3.2. Transmission Electron Microscopy (TEM) The morphology of microemulsions were investigated using transmission electron microscopy (TEM, Philips, Holland) [44, 45]. The microemulsion was stained using 0.5 % phosphotungstic acid and positioned on a carbon coated copper grid. The grid was examined under TEM to obtained point to point high resolution microemulsion images (100 kV) using different combination of bright filed imaging at increasing magnification (10,000×).
2.3.3. Thermodynamic stability studies The study investigate the effect of storage (shelf life) and shipping conditions. Centrifugation test: The microemulsions were centrifuge at 10,000 RPM for 30 minutes and observed for any phase separation [46-48]. The samples which did not showed phase separation were further tested for heating and cooling cycle. Heating-cooling cycle: The microemulsions were exposed/stored at 45oC for 48 h, followed by 4oC for 48 h [49, 50]. Such six cycles were performed. The microemulsions which did not showed any sign of phase separation were further investigated for Freeze thaw test. Freeze thaw test: The microemulsions were subjected to stress at -21oC and 25oC each for 48
h, and observed visually for phase separation or cracking or creaming [51-53]. The microemulsions which passes these tests were considered thermodynamically stable.
2.4. Fabrication of the contact lenses The contact lens was fabricated by free radical polymerization technique using monomers solution composed of siloxane (100 µL), ethyleneglycol dimethacrylate (15 µL), dimethyl acrylamide (300 µL), Irgacure® (10 mg), and hydroxyl ethylmethacrylate (HEMA, up to 1 ml) [54, 55]. The excess monomers solution was added in the female mould and male mould was carefully joined. The assembly was shifted in the Ultraviolet transilluminator and cured for 30 minutes at 350-380 nm. The contact lenses were removed and stored at 25oC (45% relative humidity) till further use.
2.5. Bimatoprost loading in the contact lenses The unreacted monomers from the fabricated contact lenses were removed by individually extracting the contact lenses in 50 ml of boiling water for 30 minutes [56-58]. The bimatoprost was loaded by soaking the contact lenses in the varying concentration of bimatoprost (25 µg/ml, 50 µg/ml and 75 µg/ml coded as SM-25, SM-50 and SM-75 respectively) in simulated tear fluid (STF, 2 ml), followed by autoclaving [59, 60]. The STF (pH 7.4) was prepared by dissolving 0.015 %w/v sodium bicarbonate and 0.9 %w/v sodium chloride in water. The concentration of the bimatoprost in the soaking solution was selected to achieve required drug loading [>30 µg, 30 µg = 5 µg × 6 dose (1 drop of 50 µl = 5 µl of 0.01 %w/v bimatoprost eye drop)] based on the preliminary studies (further discussed in section 3.3). The contact lenses were autoclaved (121oC, 15 psi for 30 minutes) in the soaking solution containing bimatoprost and thereafter soaked for a period of 7 days [61]. After soaking period, the contact lenses were removed and blotted to remove the excess of soaking solution and were subjected to evaluation studies. The selected bimatoprost-microemulsion [ME-2(1:4), based on thermodynamic stability studies] was loaded by soaking the extracted contact lenses in the STF (1.9 ml) spiked with 0.1 ml of bimatoprost-microemulsion [ME-2(1:4)] containing varying amount of 50 µg, 100 µg and 150 µg bimatoprost (in 0.1 ml), followed by autoclaving. The final
concentration of bimatoprost microemulsion in 2 ml of STF (1.9 ml STF + 0.1 ml microemulsion) was 25 µg/ml, 50 µg/ml and 75 µg/ml coded as ME-25, ME-50 and ME75 respectively. The contact lenses were sterilized (autoclaved) in the respective soaking solution containing bimatoprost-microemulsion and thereafter soaked for a period of 7 days. After soaking period, the contact lenses were removed and blotted to remove the excess of soaking solution and were subjected to evaluation studies.
2.6 Characterization of the silicone contact lenses 2.6.1. Swelling study The dry contact lenses after fabrication were carefully removed from the moulds and the weights were recorded as dry weight (W D). After 7 days of soaking, the contact lenses were removed from their respective soaking solution at room temperature, thereafter blotted using filter paper and weight (W S) again [56, 62, 63]. The percentage swelling was calculated using following formula: % Swelling =
−
× 100
2.6.2. Transmittance The transmittance of the control contact lenses (without bimatoprost), SM contact lenses and ME contact lenses were scanned using UV-vis spectrophotometer. The contact lenses were removed from the packaging solution and positioned inside the quartz cuvette, which was placed in the UV-vis spectrophotometer to measure the transmittance at 630 nm wavelength [64-66]. The experiment was conducted in triplicate.
2.6.3. Folding endurance The folding endurance test is to check the elasticity and brittleness of the soaked contact lens after 7 days of soaking in comparison to control contact lens [67-69]. The folding endurance was checked by folding the contact lens between the fingers and the thumb from the center (same place). The process was repeated till the contact lens break or visible crack was observed. The total folding operations were considered as folding endurance value.
2.7. Quantification of bimatoprost loaded in the contact lenses The uptake of bimatoprost from the soaking solutions were quantified using developed and validated HPLC method [70, 71]. The contact lenses were individually transferred in the screw capped glass tube containing 50 ml of 10 %v/v methanolic solution. The vials were agitated in the incubator shaker at 200 rpm for 30 days at 25oC to extract bimatoprost from the contact lens. After 30 days, the methanolic solution was analyzed for bimatoprost using HPLC. All readings were recorded in triplicate.
2.8. In vitro drug release study The in vitro drug release studies of the SM and ME batches were performed by placing the drug loaded contact lenses individually in 2 ml of STF media at 34oC uner constant stirring at 50 RPM in the incubator shaker [72-74]. The 2 ml aliquots of the media were withdrawn at regular time intervals for bimatoprost quantification and replaced with the same volume of the fresh STF. The sampling was carried out until there was no increase in the drug concentration in 2 successive measurements. All readings were noted in triplicate. The release profile of the drugs was evaluated by plotting different graphs like: Percentage cumulative drug release versus time, release rate (ng/h) versus time.
2.9. In vivo drug release study The New Zealand rabbits (male and female) were used to investigate the release profiles of bimatoprost in the tear fluid from the selected SM-75 contact lens (32.58 ± 5.35 µg), ME-50 (46.36 ± 5.66 µg) contact lens contrasting with the 0.03 % w/v bimatoprost eye drop (1 drop = 50 µl = 15 µg bimatoprost) [27, 75, 76]. The study protocol for white New Zealand rabbits were approved by Shandong Provincial Hospital, Shandong University (JXC20180310). The sterile contact lens was placed on the right eye (n=4) of rabbits (left eye was kept control) without local anaesthesia. In eye drop group, the rabbit’s right eye received single drop of bimatoprost eye drop (left eye was kept control). The rabbit tear fluid was collected using disposable glass capillary from the cul de sac and preserved at -20oC until analysis. The bimatoprost-tear fluid samples were treated with 1 ml of methanol to precipitate proteins, followed by freeze (5oC)
centrifugation (Remi freeze-centrifuge) for 1 h at 8000 RPM. The collected supernatant was analyzed for bimatoprost by HPLC method. One rabbit from each group was euthanized at the end of the study for histopathological analysis [77-79]. The eyes were enucleated and fixed in 10% formalin buffered solution. The paraffin embedded corneas were cut into sections using microtome and stained using hematoxylin. The structure of cornea including basement membrane, epithelium and stroma were investigated using light microscopy at ‘×400 magnification’.
2.10. Statistical analysis The statistical analysis was carried out using SPSS 20.0 for Windows. T-test (2tailed) and one-way analysis of variance was used to compare the groups.
3. Results and discussion 3.1. Characterization and selection of microemulsion Bimatoprost loaded microemulsions shows average globule size in the range of 25.65 ± 2.84 to 39.69 ± 3.69 nm (Table 2). The globule size of microemulsion increased with increase in the amount of oil phase and 1:6 ratio with same amount of Smix, which was expected. The globule size of all microemulsions batches were < 50 nm, which suggest no interference in transmittance of the contact lenses after microemulsion loading. The zeta potential was close to zero for all the microemulsion batches (Table 2), suggesting absence of any charge on the surface of oil globule due to non-ionic nature of Pluronic F68 and Tween 20. The TEM images (Supplementary material 1) showed uniformly distributed (no agglomeration) of spherical oil globules with diameters ranging from 16 to 72 nm. In thermodynamic stability studies, microemulsions were visually observed and investigated for centrifugation, heating-cooling cycles and freeze thaw cycles. The data of thermodynamic stability studies are shown in table 2. All four batches were transparent to the naked eye and did not showed any sign of creaming or cracking in the centrifugation test. Batch ME-3(1:6) and ME-4(1:6) failed in heating-cooling and freeze thaw cycles. Both the batches at 1:6 ratio showed phase separation, which was due to lower level of Pluronic F68. The results indicate, the major role of Pluronic F68 in
stabilizing microemulsion. ME-1(1:4) and ME-2(1:4) batches were thermodynamically stable. ME-2(1:4) batch of microemulsion was selected due to the presence of higher level of oil to dissolved bimatoprost in comparison to ME-1(1:4). Bimatoprost was dissolved in ME-2(1:4) microemulsion at three different concentrations (0.5 mg/ml, 1 mg/ml and 1.5 mg/ml) to use/incorporate in the soaking solution to load bimatoprost-ME in the contact lens (mentioned in section 2.5).
Table 2 Globule size, zeta potential and thermodynamic stability data of microemulsions. Globule size Zeta HeatingFreeze Visual CentrifBatches (nm) potential cooling thaw appearance ugation (mV) cycles cycles ME-1(1:4) 25.65 ± 2.84 - 2.32 ± 0.64 Transparent √ √ √ ME-2(1:4) 33.25 ± 4.25 - 2.65 ± 0.57 Transparent √ √ √ ME-3(1:6) 28.56 ± 2.41 -3.89 ± 0.74 Transparent √ X X √ X X ME-4(1:6) 39.69 ± 3.69 - 3.34 ± 0.35 Transparent 3.2. Characterization of the contact lenses 2.2.1. Swelling study The swelling report of the bimatoprost loaded contact lenses are shown in Table 3. The swelling property (water content) of the contact lens has direct relationship with the oxygen and ion permeability and final dimensions of the contact lens [80-82]. Thus, the presence of bimatoprost microemulsion should not alter the % swelling of the contact lens. The bimatoprost soaked contact lenses (SM) and bimatoprost-microemulsion soaked contact lenses (ME) did not showed significant alterations (p>0.05) in the % swelling (94.03 ± 3.33 % to 84.06 ± 3.94%) in comparison to the control contact lenses (88.86 ± 3.31 %). The bimatoprost-microemulsion soaked contact lenses (ME) showed slight increase (statistically non-significant) in the % swelling values, which was due to the presence of hydrophilic surfactants. Thus, the presence of bimatoprostmicroemulsion did not altered the swelling property, i.e. water content of the contact lenses. Thus, we expect no alterations in the oxygen and ion permeability of the bimatoprost-microemulsion contact lenses.
Table 3 Percentage swelling and transmittance data. Values are shown as mean ± standard deviations (n = 3). Transmittance p p Folding p Codes Swelling (%) (%) value value endurance value Control 99.38 ± 0.36 88.86 ± 3.31 158 ± 6 contact lens SM-25 98.62 ± 1.04 0.23 88.05 ± 2.32 0.68 157 ± 13 0.97 SM-50 98.20 ± 0.54 0.12 86.02 ± 4.10 0.53 154 ± 11 0.64 SM-75 98.15 ± 1.64 0.35 84.06 ± 3.94 0.34 151 ± 4 0.30 97.61 ± 1.00 0.11 90.43 ± 1.89 0.65 159 ± 3 0.87 ME-25 ME-50 97.57 ± 0.96 0.09 92.08 ± 1.70 0.38 151 ± 7 0.39 ME-75 96.91 ± 0.58 0.05 94.03 ± 3.33 0.06 158 ± 5 0.42 3.2.2. Transmittance study The transmittance report of the bimatoprost loaded contact lenses are shown in Table 3. The transmittance of the contact lens should be > 95 % for clear vision [83]. The bimatoprost soaked contact lenses (SM) and bimatoprost-microemulsion soaked contact lenses (ME) did not showed significant reduction (p>0.05) in the % transmittance values (98.62 ± 1.04 % to 96.91 ± 0.58 %) in comparison to the control contact lenses (99.38 ± 0.36 %). Thus, the presence of nanometric size bimatoprostmicroemulsion did not altered the transmittance of the contact lenses.
3.2.3. Folding endurance The folding endurance test indicate the elasticity and brittleness of the fabricated contact lens. The bimatoprost soaked contact lenses (SM) and bimatoprostmicroemulsion soaked contact lenses (ME) did not showed significant alterations (p>0.05, table 3) in the % transmittance values (151 ± 4 to 158 ± 5) in comparison to the control contact lenses (158 ± 6). Thus, we expect no alterations in the elasticity and the continuous polymeric matrix structure of the contact lens due to the presence of bimatoprost-microemulsion.
3.3. Quantification of bimatoprost loaded in the contact lenses The uptake/loading of bimatoprost in the contact lenses (Table 4) were quantified to investigate the uniform dispersion of bimatoprost in the contact lens and reproducibility of the methodology. The uptake of bimatoprost from the bimatoprost-soaking solution was found to be 12.38 ± 2.51 µg, 23.66 ± 3.83 µg and 32.58 ± 5.35 µg for SM-25 (25 µg/ml), SM-50 (50 µg/ml) and SM-75 (75 µg/ml) respectively, which include 6 eye drop doses [30 µg = 5 µg × 6 dose (1 drop of 50 µl = 5 µl of 0.01 %w/v bimatoprost eye drop)]. The uptake of bimatoprost from the bimatoprost-microemulsion soaking solution was found to be 26.36 ± 2.57 µg, 46.36 ± 5.66 µg and 61.39 ± 6.73 µg for ME-25, ME50 and ME-75 respectively. The standard deviation values were low enough to confirm the reproducibility of uptake of bimatoprost in the contact lenses. The data suggest approximately two fold increase in the uptake of bimatoprost from the bimatoprostmicroemulsion soaking solution in comparison to bimatoprost-soaking solution. Thus, the bimatoprost-microemulsion showed higher partition towards the contact lens matrix in comparison to the soaking solution.
Table 4 Data of bimatoprost loading from the SM and ME batches. Values are shown as mean ± standard deviations (n = 3). Bimatoprost loaded in the Batch code contact lenses (µg) SM-25 12.38 ± 2.51 SM-50 23.66 ± 3.83 SM-75 32.58 ± 5.35 ME-25 26.36 ± 2.57 ME-50 46.36 ± 5.66 ME-75 61.39 ± 6.73 3.4. In vitro drug release study The cumulative release (µg) and release rate profiles of bimatoprost from the contact lenses are shown in Fig. 1 and Fig. 2 respectively. The data of bimatoprost release from the SM batches showed cumulative release of 11.4 µg, 20.4 µg, and 32.56 µg from SM25, SM-50 and SM-75 respectively. The SM contact lenses showed low drug loading/uptake and high drug release rate (no drug detected after 48 h), making it
unsuitable technique to load bimatoprost to produce extended release therapeutic contact lens. The data of bimatoprost release from the ME batches showed cumulative release of 29.7 µg, 42.5 µg, and 59.25 µg from ME-25, ME-50 and ME-75 respectively. The ME batches showed improved uptake of bimatoprost from the bimatoprostmicroemulsion soaking solution. The release profile improved with bimatoprostmicroemulsion (ME) batches, indicated by sustained release up to 96 h. The sustained release was due to slow diffusion of bimatoprost from the tight surfactant arrangement at the oil/water interface of oil globules. The release rate at 48 h from SM-75 lenses was 77 ng/h, while ME-75 lenses shows high release rate of 33 ng/h at 96 h. The data clearly indicate the advantage of using bimatoprost-microemulsion to sustain the release rate of bimatoprost from the contact lens.
60
Cummulative release (µg)
50 SM-25 40 SM-50 30
SM-75 ME-25
20
ME-50 10 ME-75 0 0
12
24
36
48 Time (h)
60
72
84
96
Fig. 1. Cumulative release of bimatoprost from contact lenses. Values are shown as mean ± standard deviations (n=3).
SM-25 10000 SM-50
Release rate (ng/h)
SM-75 ME-25 1000
ME-50 ME-75
100
10 0
12
24
36
48 Time (h)
60
72
84
96
Fig. 2. Release rate (ng/h) of bimatoprost from the soaked contact lenses. Values are shown as mean ± standard deviations (n=3).
3.5. In vivo drug release study and histopathology reports The in vivo tear fluid analysis was performed using selected SM-75 contact lens (32.58 ± 5.35 µg), ME-50 (46.36 ± 5.66 µg) contact lens contrasting with the 0.03 % w/v bimatoprost eye drop (1 drop = 50 µl = 15 µg bimatoprost). The Cmax (5 minutes) of bimatoprost was found to be 112.25 µg/ml, 85.69 µg/ml and 154.69 µg/ml for SM-75, ME-50 and eye drop solution respectively (Fig. 3). The bimatoprost eye drop solution showed rapid fall in drug concentration (up to 1 h), while SM-75 and ME-50 contact lens showed improvement in drug retention up to 12 and 48 h respectively. ME-50 contact lens showed sustained drug release and high drug-tear fluid concentration in comparison to SM-75 contact lens. The use of ME contact lens showed reduction in the initial burst release in comparison to the eye drop solution. The hyperaemia reported with the use of eye drop solution (0.03% w/v) due to the high local bimatoprost concentration in the conjunctival tissue [84, 85] can be avoided with the use of the ME
contact lens. The corneal histopathological images of control, eye drop, SM-75 and ME50 contact lens (Fig. 4) showed normal squamous epithelium cells with normal pattern of collagen fibers in the stroma with no obvious changes. However, a long term three
Concentration of bimatoprost in tear fluid (µg/ml)
weeks data is needed to assure the safety of contact lens for clinical use. LUMIGAN® eye drop (1 drop = 15 µg bimatoprost)
100
SM-75 contact lens (32.58 ± 5.35 µg bimatoprost) ME-50 contact lens (46.36 ± 5.66 µg bimatoprost)
10
1 0
4
8
12
16
20
24 28 Time (h)
32
36
40
44
48
Fig. 3. Bimatoprost tear fluid concentration from the contact lenses and eye drop solution. Each time point represents the mean ± standard deviation (n = 4).
Fig. 4. Histopathological images of cornea, (A) control rabbit eye, (B) 0.03 % w/v bimatoprost eye drop, (C) SM-75 contact lens and (D) ME-50 contact lens.
5. Conclusion The research work explored the successful application of using bimatoprostmicroemulsion to improve the uptake of bimatoprost in the contact lenses and improvement in the drug release kinetics using soaking method. The swelling, transmittance and folding endurance values were not altered in the presence of bimatoprost microemulsion. The two fold increase in the uptake/loading of bimatoprost was observed from the bimatoprost-microemulsion (ME) soaking solution in comparison to bimatoprost-soaking solution (SM). The in vitro bimatoprost release profiles of the ME contact lenses (up to 96 h) showed improvement in the release rate profiles in comparison to SM contact lenses (up to 48 h). The in vivo studies in the rabbit tear fluid showed low burst release and improvement in the bimatoprost retention time with ME contact lens in comparison to the SM contact lens and eye drop solution. The histopathological studies of corneas showed normal squamous epithelium cells with SM contact lens in comparison to the control group. It can be concluded that the concept of using bimatoprost-loaded microemulsion improved the uptake and release kinetics of the bimatoprost from the contact lens without changing the critical contact lens properties.
Disclosures There are no potential conflicts of interest to disclose for this work.
Acknowledgments This project was funded by Key Research and Development Program of Shandong Province in China (2017GSF18178) and Shandong Provincial Natural Science Foundation, (ZR2016HB67).
Data availability Most of the raw data are available in the supplementary files. The other data could also be available on request to corresponding author.
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Conflicts of Interest To Chief Editor Subject: Letter of Submission Dear Sir/Mam, I, Jun Li declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. I wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. I confirm that the manuscript has been read and approved by all authors and that there are no other persons who satisfied the criteria for authorship. I hereby also declare that the submitted research article is original and unpublished and also is not being considered for publication elsewhere in any other journal.
Thanking you, Yours sincerely, (Dr. Guoying Mu)
S1773-2247(19)31229-8
DOI:
https://doi.org/10.1016/j.jddst.2019.101330
Reference:
JDDST 101330
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 19 August 2019 Revised Date:
14 October 2019
Accepted Date: 15 October 2019
Please cite this article as: W. Xu, W. Jiao, S. Li, X. Tao, G. Mu, Bimatoprost loaded microemulsion laden contact lens to treat glaucoma: In vitro and in vivo studies, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.101330. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
Bimatoprost loaded microemulsion laden contact lens to treat glaucoma: In vitro and in vivo studies Wenwen Xu a, Wanzhen Jiao a, Shangbin Li b, Xiangchen Tao a, Guoying Mu * a a
Department of Ophthalmology, Shandong Provincial Hospital, Shandong University,
Shandong 250021, China b
Department of Healthcare, Shandong Provincial Hospital, Shandong University,
Shandong 250021,China
* Corresponding author at: Department of Ophthalmology, Shandong Provincial Hospital, Shandong University, Jingwu Road, Huaiyin District, Jinan, Shandong 250021, China. Tel.: +861 8732200909 E-mail address: [email protected] (G. Mu)
Abstract Bimatoprost is widely used to manage glaucoma. Currently, it is delivered via eye drop solution in high doses due to poor ocular bioavailability. The high and daily ocular dosing lead to ocular hyperaemia causing patient non-compliance. The contact lenses can be used to sustain the release of drug by conventional soaking technology, without altering the critical contact lens properties. However, the soaking method showed low drug uptake and high burst release, due to the absence of efficient controlling membrane. Here we investigate the effect of microemulsion on bimatoprost uptake from the soaking solution and its effect on drug release kinetics. The contact lenses were soaked in bimatoprost-microemulsion soaking solution (ME) and compared with the bimatoprost-soaking solution (SM) without microemulsion. The uptake of bimatoprostmicroemulsion in the contact lenses did not altered the swelling, transmittance and folding endurance properties. The two fold increase in the uptake/loading of bimatoprost was noted from the bimatoprost-microemulsion (ME) soaking solution in comparison to bimatoprost-soaking solution (SM). The in vitro flux data of ME batches (up to 96h) showed improvement in the release rate profiles in comparison to SM batches (up to 48h). The in vivo studies in the rabbit tear fluid showed low burst release and improvement in the bimatoprost retention time with ME contact lens in comparison to the SM contact lens and eye drop solution. The study demonstrate the potential of microemulsion to improve the uptake of bimatoprost and sustain release kinetics without altering the critical lens properties of the contact lens.
Key words: Bimatoprost, silicone contact lenses, soaking method, microemulsion, animal studies.
1. Introduction Bimatoprost is widely used to manage glaucoma by the eye drop solution in high doses (1 drop ≈ 15 µg, 0.03%w/v Lumigan eye drop), due to short ocular drug residence time and poor ocular bioavailability [1-5]. The daily high ocular dosing lead to ocular hyperaemia causing patient non-compliance [6-9]. The silicon contact lenses can be used to sustain the release of bimatoprost using soaking technique without affecting the critical contact lens properties [10-12]. However, the soaking method showed low drug uptake and high burst release, due to the absence of efficient controlling membrane [1316]. Thus, an innovative approach is desirable to increase the drug uptake along with sustain release capacity of the contact lens. Several development strategies have been reported to sustain the release of ocular drugs from the contact lenses including soaking method [17-19], polymeric nanoparticles [20-22], molecular imprinting [23-25], PLGA films [26, 27], implantation technology [28, 29], use of supercritical fluids [30], etc. The strategies do showed promising application of the contact lenses, however, they failed in the optimal clinical translation due to alteration in the critical contact lens properties. The soaking method is widely applied to load ophthalmic drugs in the contact lenses. C. M. Phan et al., 2016 loaded moxifloxacin in the silicon contact lenses by soaking method and observed high burst release [31]. C. Chung et al., 2005 developed timolol loaded contact lenses by soaking method, which showed improved drug residence time with low dose [32]. J. Hui et al., 2013 design ciprofloxacin and dexamethasone loaded contact lenses by soaking method using cyclodextrin, which showed high burst release [33]. Andreza et al., 2012 loaded acetazolamide using cyclodextrin pendant contact lens, the system shoed 12 h release [34]. J. Xu et al., 2010 loaded puerarin using β-cyclodextrin in hydrogel to treat glaucoma. The contact lens showed alteration in the swelling and tensile strength with high burst release in flux studies [35]. K. Hui et al., 2015 loaded timolol and dorzolamide by soaking method using Vitamin E contact lens. The system showed 48 h reduction in the IOP in Beagle dogs [36]. C-Chung et al., 2007 used microemulsion to load timolol in the contact lens, which showed sustain release for days [37]. The drug loading by the conventional soaking method does not alter the critical lens properties [38]; however, it
pose the issue of low drug loading and high burst release [14], which is not ideal for the sustain drug delivery. The objective of this paper was to address the issues of conventional soaking technique using microemulsion. The study investigate the effect of bimatoprost loading from the soaking solution and its effect on the drug release kinetics from the contact lens. The contact lenses were soaked in bimatoprost-microemulsion soaking solution and compared with the bimatoprost-soaking solution without microemulsion. The bimatoprost loaded contact lenses were evaluated for in vitro and in vivo studies; to investigate the effect of microemulsion on the bimatoprost uptake and its release kinetics from the contact lenses.
2. Materials and Methods 2.1 Materials Hydroxyl ethylmethacrylate (HEMA), ethyleneglycol dimethacrylate (EGDMA), Irgacure®, dimethyl acrylamide (DMA), and Siloxane (TRIS) were purchased from Sigma-Aldrich Chemicals (MO, USA). Bimatoprost was purchased from Guangzhou Quanao Chemical Co., Ltd. (Guangdong, China). All other reagents and chemicals were purchased from Sigma-Aldrich Chemicals (MO, USA).
2.2 Preparation of microemulsion The microemulsion batches were selected from the pseudo ternary phase diagrams [Smix (1:4 and 1:6) ratio] (supplementary material 1) and the composition are shown in table 1. The Smix solution (Pluronic F68 and Tween 20) was dissolved in required volume of water with constant stirring at room temperature. The required quantity of isopropyl myristate was added drop by drop at 50 RPM to get clear transparent microemulsion [39-41]. One batch of microemulsion was selected to load bimatoprost for further studies based on thermodynamic stability results (section 3.1). Bimatoprost was dissolved in the selected microemulsion at three different levels (0.5 mg/ml, 1 mg/ml and 1.5 mg/ml) to use/incorporate in the soaking solution to load bimatoprost in the contact lens (see section 2.5).
Table 1 Composition and coding of microemulsion. Water (%) Codes Isopropyl myristate (%) Smix solution (%) ME-1(1:4) 1.5 50 48.5 ME-2(1:4) 3 50 47 ME-3(1:6) 1.5 50 48.5 ME-4(1:6) 3 50 47 Smix = Pluronic F68: Tween 20, ME = microemulsion, 1:4 and 1:6 are Smix ratios 2.3. Characterization of microemulsions 2.3.1. Globule size and zeta potential The globule size and zeta potential of microemulsions were determined by Malvern Zetasizer (3000HSA, UK) at 37oC. Sample microemulsion was placed in standard glass cuvette and size was determined at 90o incidence angle [42, 43]. Reading was recorded in triplicate.
2.3.2. Transmission Electron Microscopy (TEM) The morphology of microemulsions were investigated using transmission electron microscopy (TEM, Philips, Holland) [44, 45]. The microemulsion was stained using 0.5 % phosphotungstic acid and positioned on a carbon coated copper grid. The grid was examined under TEM to obtained point to point high resolution microemulsion images (100 kV) using different combination of bright filed imaging at increasing magnification (10,000×).
2.3.3. Thermodynamic stability studies The study investigate the effect of storage (shelf life) and shipping conditions. Centrifugation test: The microemulsions were centrifuge at 10,000 RPM for 30 minutes and observed for any phase separation [46-48]. The samples which did not showed phase separation were further tested for heating and cooling cycle. Heating-cooling cycle: The microemulsions were exposed/stored at 45oC for 48 h, followed by 4oC for 48 h [49, 50]. Such six cycles were performed. The microemulsions which did not showed any sign of phase separation were further investigated for Freeze thaw test. Freeze thaw test: The microemulsions were subjected to stress at -21oC and 25oC each for 48
h, and observed visually for phase separation or cracking or creaming [51-53]. The microemulsions which passes these tests were considered thermodynamically stable.
2.4. Fabrication of the contact lenses The contact lens was fabricated by free radical polymerization technique using monomers solution composed of siloxane (100 µL), ethyleneglycol dimethacrylate (15 µL), dimethyl acrylamide (300 µL), Irgacure® (10 mg), and hydroxyl ethylmethacrylate (HEMA, up to 1 ml) [54, 55]. The excess monomers solution was added in the female mould and male mould was carefully joined. The assembly was shifted in the Ultraviolet transilluminator and cured for 30 minutes at 350-380 nm. The contact lenses were removed and stored at 25oC (45% relative humidity) till further use.
2.5. Bimatoprost loading in the contact lenses The unreacted monomers from the fabricated contact lenses were removed by individually extracting the contact lenses in 50 ml of boiling water for 30 minutes [56-58]. The bimatoprost was loaded by soaking the contact lenses in the varying concentration of bimatoprost (25 µg/ml, 50 µg/ml and 75 µg/ml coded as SM-25, SM-50 and SM-75 respectively) in simulated tear fluid (STF, 2 ml), followed by autoclaving [59, 60]. The STF (pH 7.4) was prepared by dissolving 0.015 %w/v sodium bicarbonate and 0.9 %w/v sodium chloride in water. The concentration of the bimatoprost in the soaking solution was selected to achieve required drug loading [>30 µg, 30 µg = 5 µg × 6 dose (1 drop of 50 µl = 5 µl of 0.01 %w/v bimatoprost eye drop)] based on the preliminary studies (further discussed in section 3.3). The contact lenses were autoclaved (121oC, 15 psi for 30 minutes) in the soaking solution containing bimatoprost and thereafter soaked for a period of 7 days [61]. After soaking period, the contact lenses were removed and blotted to remove the excess of soaking solution and were subjected to evaluation studies. The selected bimatoprost-microemulsion [ME-2(1:4), based on thermodynamic stability studies] was loaded by soaking the extracted contact lenses in the STF (1.9 ml) spiked with 0.1 ml of bimatoprost-microemulsion [ME-2(1:4)] containing varying amount of 50 µg, 100 µg and 150 µg bimatoprost (in 0.1 ml), followed by autoclaving. The final
concentration of bimatoprost microemulsion in 2 ml of STF (1.9 ml STF + 0.1 ml microemulsion) was 25 µg/ml, 50 µg/ml and 75 µg/ml coded as ME-25, ME-50 and ME75 respectively. The contact lenses were sterilized (autoclaved) in the respective soaking solution containing bimatoprost-microemulsion and thereafter soaked for a period of 7 days. After soaking period, the contact lenses were removed and blotted to remove the excess of soaking solution and were subjected to evaluation studies.
2.6 Characterization of the silicone contact lenses 2.6.1. Swelling study The dry contact lenses after fabrication were carefully removed from the moulds and the weights were recorded as dry weight (W D). After 7 days of soaking, the contact lenses were removed from their respective soaking solution at room temperature, thereafter blotted using filter paper and weight (W S) again [56, 62, 63]. The percentage swelling was calculated using following formula: % Swelling =
−
× 100
2.6.2. Transmittance The transmittance of the control contact lenses (without bimatoprost), SM contact lenses and ME contact lenses were scanned using UV-vis spectrophotometer. The contact lenses were removed from the packaging solution and positioned inside the quartz cuvette, which was placed in the UV-vis spectrophotometer to measure the transmittance at 630 nm wavelength [64-66]. The experiment was conducted in triplicate.
2.6.3. Folding endurance The folding endurance test is to check the elasticity and brittleness of the soaked contact lens after 7 days of soaking in comparison to control contact lens [67-69]. The folding endurance was checked by folding the contact lens between the fingers and the thumb from the center (same place). The process was repeated till the contact lens break or visible crack was observed. The total folding operations were considered as folding endurance value.
2.7. Quantification of bimatoprost loaded in the contact lenses The uptake of bimatoprost from the soaking solutions were quantified using developed and validated HPLC method [70, 71]. The contact lenses were individually transferred in the screw capped glass tube containing 50 ml of 10 %v/v methanolic solution. The vials were agitated in the incubator shaker at 200 rpm for 30 days at 25oC to extract bimatoprost from the contact lens. After 30 days, the methanolic solution was analyzed for bimatoprost using HPLC. All readings were recorded in triplicate.
2.8. In vitro drug release study The in vitro drug release studies of the SM and ME batches were performed by placing the drug loaded contact lenses individually in 2 ml of STF media at 34oC uner constant stirring at 50 RPM in the incubator shaker [72-74]. The 2 ml aliquots of the media were withdrawn at regular time intervals for bimatoprost quantification and replaced with the same volume of the fresh STF. The sampling was carried out until there was no increase in the drug concentration in 2 successive measurements. All readings were noted in triplicate. The release profile of the drugs was evaluated by plotting different graphs like: Percentage cumulative drug release versus time, release rate (ng/h) versus time.
2.9. In vivo drug release study The New Zealand rabbits (male and female) were used to investigate the release profiles of bimatoprost in the tear fluid from the selected SM-75 contact lens (32.58 ± 5.35 µg), ME-50 (46.36 ± 5.66 µg) contact lens contrasting with the 0.03 % w/v bimatoprost eye drop (1 drop = 50 µl = 15 µg bimatoprost) [27, 75, 76]. The study protocol for white New Zealand rabbits were approved by Shandong Provincial Hospital, Shandong University (JXC20180310). The sterile contact lens was placed on the right eye (n=4) of rabbits (left eye was kept control) without local anaesthesia. In eye drop group, the rabbit’s right eye received single drop of bimatoprost eye drop (left eye was kept control). The rabbit tear fluid was collected using disposable glass capillary from the cul de sac and preserved at -20oC until analysis. The bimatoprost-tear fluid samples were treated with 1 ml of methanol to precipitate proteins, followed by freeze (5oC)
centrifugation (Remi freeze-centrifuge) for 1 h at 8000 RPM. The collected supernatant was analyzed for bimatoprost by HPLC method. One rabbit from each group was euthanized at the end of the study for histopathological analysis [77-79]. The eyes were enucleated and fixed in 10% formalin buffered solution. The paraffin embedded corneas were cut into sections using microtome and stained using hematoxylin. The structure of cornea including basement membrane, epithelium and stroma were investigated using light microscopy at ‘×400 magnification’.
2.10. Statistical analysis The statistical analysis was carried out using SPSS 20.0 for Windows. T-test (2tailed) and one-way analysis of variance was used to compare the groups.
3. Results and discussion 3.1. Characterization and selection of microemulsion Bimatoprost loaded microemulsions shows average globule size in the range of 25.65 ± 2.84 to 39.69 ± 3.69 nm (Table 2). The globule size of microemulsion increased with increase in the amount of oil phase and 1:6 ratio with same amount of Smix, which was expected. The globule size of all microemulsions batches were < 50 nm, which suggest no interference in transmittance of the contact lenses after microemulsion loading. The zeta potential was close to zero for all the microemulsion batches (Table 2), suggesting absence of any charge on the surface of oil globule due to non-ionic nature of Pluronic F68 and Tween 20. The TEM images (Supplementary material 1) showed uniformly distributed (no agglomeration) of spherical oil globules with diameters ranging from 16 to 72 nm. In thermodynamic stability studies, microemulsions were visually observed and investigated for centrifugation, heating-cooling cycles and freeze thaw cycles. The data of thermodynamic stability studies are shown in table 2. All four batches were transparent to the naked eye and did not showed any sign of creaming or cracking in the centrifugation test. Batch ME-3(1:6) and ME-4(1:6) failed in heating-cooling and freeze thaw cycles. Both the batches at 1:6 ratio showed phase separation, which was due to lower level of Pluronic F68. The results indicate, the major role of Pluronic F68 in
stabilizing microemulsion. ME-1(1:4) and ME-2(1:4) batches were thermodynamically stable. ME-2(1:4) batch of microemulsion was selected due to the presence of higher level of oil to dissolved bimatoprost in comparison to ME-1(1:4). Bimatoprost was dissolved in ME-2(1:4) microemulsion at three different concentrations (0.5 mg/ml, 1 mg/ml and 1.5 mg/ml) to use/incorporate in the soaking solution to load bimatoprost-ME in the contact lens (mentioned in section 2.5).
Table 2 Globule size, zeta potential and thermodynamic stability data of microemulsions. Globule size Zeta HeatingFreeze Visual CentrifBatches (nm) potential cooling thaw appearance ugation (mV) cycles cycles ME-1(1:4) 25.65 ± 2.84 - 2.32 ± 0.64 Transparent √ √ √ ME-2(1:4) 33.25 ± 4.25 - 2.65 ± 0.57 Transparent √ √ √ ME-3(1:6) 28.56 ± 2.41 -3.89 ± 0.74 Transparent √ X X √ X X ME-4(1:6) 39.69 ± 3.69 - 3.34 ± 0.35 Transparent 3.2. Characterization of the contact lenses 2.2.1. Swelling study The swelling report of the bimatoprost loaded contact lenses are shown in Table 3. The swelling property (water content) of the contact lens has direct relationship with the oxygen and ion permeability and final dimensions of the contact lens [80-82]. Thus, the presence of bimatoprost microemulsion should not alter the % swelling of the contact lens. The bimatoprost soaked contact lenses (SM) and bimatoprost-microemulsion soaked contact lenses (ME) did not showed significant alterations (p>0.05) in the % swelling (94.03 ± 3.33 % to 84.06 ± 3.94%) in comparison to the control contact lenses (88.86 ± 3.31 %). The bimatoprost-microemulsion soaked contact lenses (ME) showed slight increase (statistically non-significant) in the % swelling values, which was due to the presence of hydrophilic surfactants. Thus, the presence of bimatoprostmicroemulsion did not altered the swelling property, i.e. water content of the contact lenses. Thus, we expect no alterations in the oxygen and ion permeability of the bimatoprost-microemulsion contact lenses.
Table 3 Percentage swelling and transmittance data. Values are shown as mean ± standard deviations (n = 3). Transmittance p p Folding p Codes Swelling (%) (%) value value endurance value Control 99.38 ± 0.36 88.86 ± 3.31 158 ± 6 contact lens SM-25 98.62 ± 1.04 0.23 88.05 ± 2.32 0.68 157 ± 13 0.97 SM-50 98.20 ± 0.54 0.12 86.02 ± 4.10 0.53 154 ± 11 0.64 SM-75 98.15 ± 1.64 0.35 84.06 ± 3.94 0.34 151 ± 4 0.30 97.61 ± 1.00 0.11 90.43 ± 1.89 0.65 159 ± 3 0.87 ME-25 ME-50 97.57 ± 0.96 0.09 92.08 ± 1.70 0.38 151 ± 7 0.39 ME-75 96.91 ± 0.58 0.05 94.03 ± 3.33 0.06 158 ± 5 0.42 3.2.2. Transmittance study The transmittance report of the bimatoprost loaded contact lenses are shown in Table 3. The transmittance of the contact lens should be > 95 % for clear vision [83]. The bimatoprost soaked contact lenses (SM) and bimatoprost-microemulsion soaked contact lenses (ME) did not showed significant reduction (p>0.05) in the % transmittance values (98.62 ± 1.04 % to 96.91 ± 0.58 %) in comparison to the control contact lenses (99.38 ± 0.36 %). Thus, the presence of nanometric size bimatoprostmicroemulsion did not altered the transmittance of the contact lenses.
3.2.3. Folding endurance The folding endurance test indicate the elasticity and brittleness of the fabricated contact lens. The bimatoprost soaked contact lenses (SM) and bimatoprostmicroemulsion soaked contact lenses (ME) did not showed significant alterations (p>0.05, table 3) in the % transmittance values (151 ± 4 to 158 ± 5) in comparison to the control contact lenses (158 ± 6). Thus, we expect no alterations in the elasticity and the continuous polymeric matrix structure of the contact lens due to the presence of bimatoprost-microemulsion.
3.3. Quantification of bimatoprost loaded in the contact lenses The uptake/loading of bimatoprost in the contact lenses (Table 4) were quantified to investigate the uniform dispersion of bimatoprost in the contact lens and reproducibility of the methodology. The uptake of bimatoprost from the bimatoprost-soaking solution was found to be 12.38 ± 2.51 µg, 23.66 ± 3.83 µg and 32.58 ± 5.35 µg for SM-25 (25 µg/ml), SM-50 (50 µg/ml) and SM-75 (75 µg/ml) respectively, which include 6 eye drop doses [30 µg = 5 µg × 6 dose (1 drop of 50 µl = 5 µl of 0.01 %w/v bimatoprost eye drop)]. The uptake of bimatoprost from the bimatoprost-microemulsion soaking solution was found to be 26.36 ± 2.57 µg, 46.36 ± 5.66 µg and 61.39 ± 6.73 µg for ME-25, ME50 and ME-75 respectively. The standard deviation values were low enough to confirm the reproducibility of uptake of bimatoprost in the contact lenses. The data suggest approximately two fold increase in the uptake of bimatoprost from the bimatoprostmicroemulsion soaking solution in comparison to bimatoprost-soaking solution. Thus, the bimatoprost-microemulsion showed higher partition towards the contact lens matrix in comparison to the soaking solution.
Table 4 Data of bimatoprost loading from the SM and ME batches. Values are shown as mean ± standard deviations (n = 3). Bimatoprost loaded in the Batch code contact lenses (µg) SM-25 12.38 ± 2.51 SM-50 23.66 ± 3.83 SM-75 32.58 ± 5.35 ME-25 26.36 ± 2.57 ME-50 46.36 ± 5.66 ME-75 61.39 ± 6.73 3.4. In vitro drug release study The cumulative release (µg) and release rate profiles of bimatoprost from the contact lenses are shown in Fig. 1 and Fig. 2 respectively. The data of bimatoprost release from the SM batches showed cumulative release of 11.4 µg, 20.4 µg, and 32.56 µg from SM25, SM-50 and SM-75 respectively. The SM contact lenses showed low drug loading/uptake and high drug release rate (no drug detected after 48 h), making it
unsuitable technique to load bimatoprost to produce extended release therapeutic contact lens. The data of bimatoprost release from the ME batches showed cumulative release of 29.7 µg, 42.5 µg, and 59.25 µg from ME-25, ME-50 and ME-75 respectively. The ME batches showed improved uptake of bimatoprost from the bimatoprostmicroemulsion soaking solution. The release profile improved with bimatoprostmicroemulsion (ME) batches, indicated by sustained release up to 96 h. The sustained release was due to slow diffusion of bimatoprost from the tight surfactant arrangement at the oil/water interface of oil globules. The release rate at 48 h from SM-75 lenses was 77 ng/h, while ME-75 lenses shows high release rate of 33 ng/h at 96 h. The data clearly indicate the advantage of using bimatoprost-microemulsion to sustain the release rate of bimatoprost from the contact lens.
60
Cummulative release (µg)
50 SM-25 40 SM-50 30
SM-75 ME-25
20
ME-50 10 ME-75 0 0
12
24
36
48 Time (h)
60
72
84
96
Fig. 1. Cumulative release of bimatoprost from contact lenses. Values are shown as mean ± standard deviations (n=3).
SM-25 10000 SM-50
Release rate (ng/h)
SM-75 ME-25 1000
ME-50 ME-75
100
10 0
12
24
36
48 Time (h)
60
72
84
96
Fig. 2. Release rate (ng/h) of bimatoprost from the soaked contact lenses. Values are shown as mean ± standard deviations (n=3).
3.5. In vivo drug release study and histopathology reports The in vivo tear fluid analysis was performed using selected SM-75 contact lens (32.58 ± 5.35 µg), ME-50 (46.36 ± 5.66 µg) contact lens contrasting with the 0.03 % w/v bimatoprost eye drop (1 drop = 50 µl = 15 µg bimatoprost). The Cmax (5 minutes) of bimatoprost was found to be 112.25 µg/ml, 85.69 µg/ml and 154.69 µg/ml for SM-75, ME-50 and eye drop solution respectively (Fig. 3). The bimatoprost eye drop solution showed rapid fall in drug concentration (up to 1 h), while SM-75 and ME-50 contact lens showed improvement in drug retention up to 12 and 48 h respectively. ME-50 contact lens showed sustained drug release and high drug-tear fluid concentration in comparison to SM-75 contact lens. The use of ME contact lens showed reduction in the initial burst release in comparison to the eye drop solution. The hyperaemia reported with the use of eye drop solution (0.03% w/v) due to the high local bimatoprost concentration in the conjunctival tissue [84, 85] can be avoided with the use of the ME
contact lens. The corneal histopathological images of control, eye drop, SM-75 and ME50 contact lens (Fig. 4) showed normal squamous epithelium cells with normal pattern of collagen fibers in the stroma with no obvious changes. However, a long term three
Concentration of bimatoprost in tear fluid (µg/ml)
weeks data is needed to assure the safety of contact lens for clinical use. LUMIGAN® eye drop (1 drop = 15 µg bimatoprost)
100
SM-75 contact lens (32.58 ± 5.35 µg bimatoprost) ME-50 contact lens (46.36 ± 5.66 µg bimatoprost)
10
1 0
4
8
12
16
20
24 28 Time (h)
32
36
40
44
48
Fig. 3. Bimatoprost tear fluid concentration from the contact lenses and eye drop solution. Each time point represents the mean ± standard deviation (n = 4).
Fig. 4. Histopathological images of cornea, (A) control rabbit eye, (B) 0.03 % w/v bimatoprost eye drop, (C) SM-75 contact lens and (D) ME-50 contact lens.
5. Conclusion The research work explored the successful application of using bimatoprostmicroemulsion to improve the uptake of bimatoprost in the contact lenses and improvement in the drug release kinetics using soaking method. The swelling, transmittance and folding endurance values were not altered in the presence of bimatoprost microemulsion. The two fold increase in the uptake/loading of bimatoprost was observed from the bimatoprost-microemulsion (ME) soaking solution in comparison to bimatoprost-soaking solution (SM). The in vitro bimatoprost release profiles of the ME contact lenses (up to 96 h) showed improvement in the release rate profiles in comparison to SM contact lenses (up to 48 h). The in vivo studies in the rabbit tear fluid showed low burst release and improvement in the bimatoprost retention time with ME contact lens in comparison to the SM contact lens and eye drop solution. The histopathological studies of corneas showed normal squamous epithelium cells with SM contact lens in comparison to the control group. It can be concluded that the concept of using bimatoprost-loaded microemulsion improved the uptake and release kinetics of the bimatoprost from the contact lens without changing the critical contact lens properties.
Disclosures There are no potential conflicts of interest to disclose for this work.
Acknowledgments This project was funded by Key Research and Development Program of Shandong Province in China (2017GSF18178) and Shandong Provincial Natural Science Foundation, (ZR2016HB67).
Data availability Most of the raw data are available in the supplementary files. The other data could also be available on request to corresponding author.
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Conflicts of Interest To Chief Editor Subject: Letter of Submission Dear Sir/Mam, I, Jun Li declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. I wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. I confirm that the manuscript has been read and approved by all authors and that there are no other persons who satisfied the criteria for authorship. I hereby also declare that the submitted research article is original and unpublished and also is not being considered for publication elsewhere in any other journal.
Thanking you, Yours sincerely, (Dr. Guoying Mu)