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In vitro evaluation of enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes
European Journal of Pharmacology 705 (2013) 20–25
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
In vitro evaluation of enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes Hui-Ping Qi a,f,1, Xiang-Chun Gao b,1, Li-Qiong Zhang a, Shu-Qin Wei c, Sheng Bi d,e, Zi-Chao Yang f, Hao Cui a,n a
Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, China Department of Ophthalmology, Harbin Medical University, Harbin, China Perinatal Epidemiology, CHU Sainte-Justine University of Montreal, Montreal, Canada d Department of Neurology, the First Affiliated Hospital of Harbin Medical University, China e Central Laboratory, the First Affiliated Hospital of Harbin Medical University, China f Department of Neurology, the Fourth Affiliated Hospital of Harbin Medical University, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 August 2012 Received in revised form 8 February 2013 Accepted 14 February 2013 Available online 28 February 2013
To investigate the enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes. Six compounds, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa were selected as model drugs. Permeation studies were performed using excised cornea of rabbits by a Franz-type diffusion apparatus. The safety of borneol was assessed on the basis of corneal hydration level and Draize eye test. The application of 0.2% borneol to the cornea increased the apparent permeability coefficient by 1.82—(P o 0.05), 2.49—(P o0.05), 4.18—(P o 0.05) and 1.11-fold (not significant) for rhodamine B, sodium-fluorescein, FITC-dextrans of 4 and 10 kDa, respectively. No significant permeability enhancement of FITC dextrans of 10, 20 and 40 kDa with borneol was found compared to control. The permeability coefficient enhanced by 0.2% borneol was linear correlated to the molecular weight of model drugs (R2 ¼ 0.9976). With the 0.05%, 0.1% and 0.2% borneol application, the corneal hydration values were o83% and Draize scores were o 4. Borneol may improve the transcorneal penetration of both hydrophilic and lipophilic compounds without causing toxic reactions, especially hydrophilic ones. Furthermore, 0.2% borneol can enhance the permeation of hydrophilic compounds with molecular weight r 4 kDa. Hence, borneol can be considered as a safe and effective penetration enhancer for ocular drug administration. & 2013 Elsevier B.V. All rights reserved.
Keywords: Permeability enhancers Borneol Transcorneal permeation Ocular irritation
1. Introduction Most ophthalmic drugs are administrated topically in the form of eye drops due to its convenience and safety (Eljarrat-Binstock et al., 2010; Rawas-Qalaji and Williams, 2012). The cornea consisted of three primary layers, the epithelium, stroma and endothelium, is generally recognized as the major route of ocular penetration for topically instilled drugs (Robinson, 1993). The lipophilic corneal epithelium contains 5–7 layers of cells each connected by tight junctions and restricts the diffusion of most hydrophilic drugs through the paracellular pathway; while the stroma, which is mainly composed of hydrated collagen, exerts a
n Correspondence to: Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, 23rd Youzheng Street, Nangang District, Harbin, Heilongjiang Province 150001, China. E-mail address: [email protected] (H. Cui). 1 Hui-Ping Qi and Xiang-Chun Gao contributed equally to this work.
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.02.031
diffusional barrier to highly lipophilic drugs through the transcellular pathway (Robinson, 1993; Salama et al., 2006; Bucolo et al., 2012). Though the endothelium is not a significant barrier to the transcorneal diffusion, its permeability depends on molecular weight rather than characteristic of compound (Bucolo et al., 2012). Therefore, it is quite difficult to ensure a successful delivery of drugs to the intended area in the eye through local application. The ocular bioavailability is very low, typically less than 5% (Eljarrat-Binstock et al., 2010; Sahoo et al., 2008; Davies, 2000). Various attempts have been made to improve drug bioavailability by increasing corneal drug penetration. An alternative approach is to transiently increase the corneal permeation with adding appropriate penetration enhancers to drug formulas. An ideal penetration enhancer is nontoxic, nonirritant, chemically and pharmacologically inert, and compatible with formulation ingredients (Liu et al., 2011). In addition, it should have a fast and reversible onset of action and should be effective at low concentrations (Liu et al., 2011). So far, many enhancers, such as
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fluorescein and rhodamine B were obtained from Melone Pharmaceutical Co., Ltd. (Dalian, China). Other chemicals were of reagent grade and obtained from Hengxing Pure Chemical Co., Ltd. (Tianjin, China). 2.2. Animals
Fig. 1. Chemical structure of borneol.
benzalkonium chloride, ethylenediamine tetra-acetic acid (EDTA), nonionic surfactants, surface-active heteroglycosides, bile salts, polycarbophil–cysteine conjugates and boric acid have been used (Kaur and Smitha, 2002). However, some of them cause significant ocular irritation or toxicity even at low concentrations. Borneol (C10H18O, molecular weight, 154.24) is a naturallyoccurring cyclic terpene alcohol extracted from the resin and volatile oil of dipterocarp (Fig. 1). It has been extensively used in pharmaceutical preparation of ophthalmic ointment, topical drops and Chinese herbal drugs in China and other Asian countries (Li et al., 2010). Some studies showed that borneol can act as a penetration enhancer for a number of drugs administered through various physiologic barriers such as skin (Cui et al., 2011), brain (Cai et al., 2008), and mucous membrane (Lu et al., 2009). Recently, several studies indicated that borneol has the potential to be used as an ocular penetration enhancer (Li et al., 2010; Yang et al., 2009; Wu et al., 2006). Wu et al. (2006) reported that borneol enhanced permeation of puerarin through the isolated cornea but did not enhance the transcorneal permeation of maleate. Yang et al. (2009) showed that borneol could promote the corneal permeability of two lipophilic (indometacin and dexamethasone) and three hydrophilic drugs (ofloxacin, tobramycin and virazle) in vitro. Li et al. (2010) showed that borneol could enhance the diffusion of danshensu across the ocular–blood barrier. Jin et al. (2011) also reported that it can reversibly open blood–optic nerve barrier without causing any damage to the blood–optic nerve barrier. As we all know, patient compliance and comfort considerations in drug administration may impact the drug’s therapeutic efficacy. Compared to other penetration enhancers, the major advantages of borneol are the wide use for ocular disease and the long term clinical safety. Currently, studies reported that borneol could enhance corneal permeability for drugs with low molecular weight. More compounds with different molecular weights, especially high molecular weight, were not tested. Further studies are still essential to verify the penetration-enhancing effect of borneol on ocular absorption and to explore the relevant mechanisms. The present study was designed to explore the transcorneal permeation enhancing effect of borneol in vitro on a variety of compounds with different hydrophilicities and molecular sizes, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa. Potential toxicity produced by borneol was assessed in vitro by measuring corneal hydration levels and in vivo by Draize eye test.
2. Materials and methods 2.1. Materials Borneol, FITC dextrans of 4, 10, 20 and 40 kDa were obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, U.S.A.). Sodium-
New Zealand white rabbits weighing 2.5–3.0 kg were provided by Institute of Experimental Animals, Harbin Medical University. The animals were given food and water ad libitum. The animal room was well ventilated, and a regular 12 h:12 h light–dark cycle was maintained throughout the experimental period. Animal care and protocols were in accordance with institutional guidelines and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Research. 2.3. In vitro transcorneal permeation studies The transcorneal permeation studies were evaluated across excised rabbit corneas. In brief, the rabbits were sacrificed by injecting air into the marginal ear vein. The cornea was dissected with a scleral ring (5 mm laterally from the limbus), and the lens and iris were carefully removed. Freshly excised rabbit corneas were immediately mounted over the modified Franz-type vertical diffusion chambers. Blank glutathione bicarbonate ringer (GBR) buffer (9 ml, pH 7.4) was added to the endothelial side and 1 ml of donor solution of the compounds with various concentrations of borneol (0%, 0.05%, 0.1%, and 0.2%, w/v) was added to the epithelial side. Borneol was dissolved in the GBR buffer containing 4% propylene glycol and 1% tween-80 as cosolvent. Sodiumfluorescein (molecular weight, 376) and rhodamine B (molecular weight, 479) were both at a concentration of 150 mg/ml, and FITCdextrans of 4, 10, 20 and 40 kDa were at a concentration of 1 mg/ ml. The solutions in each chamber were mixed by bubbling an O2–CO2 (95:5) mixture at the rate of three to four bubbles per second and maintaining the temperature at 35 71 1C under mixing conditions using a constant magnetic stirring with a rotating speed of 600 rpm. The corneal area available for diffusion was 0.785 cm2. At intervals of 30 min, until 240 min, samples of 200 ml were taken from the endothelial side and replaced by an equal volume of blank GBR buffer. Concentrations of sodiumfluorescein, FITC-dextrans of 4, 10, 20 and 40 kDa were determined using a multi-mode microplate reader (SpectraMax M5, Molecular Devices, USA) with 485-nm excitation and 530-nm emission filters. Concentrations of rhodamine B were determined by the multi-mode microplate reader with 530-nm excitation and 590-nm emission filters. All the experiments were conducted in four repeats. The corneal permeation parameters were calculated from the plot of cumulative amount of compounds permeated as a function of time. The accumulated amounts of compounds permeated across the cornea were calculated by the following Eq. (1): ! n1 n1 X V X Q ¼ V 0 Cn þ Ci ¼ V 0Cn þ V Ci ð1Þ V0 i ¼ 1 i¼1 Q—the accumulated permeation amount within time t, Cn—the measured concentration value within time t, V0—the total volume of solution in the reservoir chamber, V—the sampling volume per time point, Ci—the measured value of concentration before time t. The apparent permeability coefficient (Papp) and steady-state flux (Jss) of the compounds were calculated by the following Eqs. (2) and (3), assuming passive diffusion under steady-state conditions: Papp ¼
DQ Dt C 0 A 60
ð2Þ
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J ss ¼ C 0 P app
ð3Þ
where DQ/Dt is the steady-state slope of the linear portion of the cumulative permeated amount of drug in the receiving chamber (Q) versus time (t), C0 is the initial concentration of the compound dissolved in the epithelial donor chamber, A is the exposed corneal surface area (0.785 cm2), and 60 is the factor for conversion of minute to second. The lag time calculated for each plot was the time axis intercept of the regression line. The Papp increase was measured by the enhancement ratio, calculated as the ratio between the Papp values obtained in the absence and presence of borneol.
The corneal opacity was graded on a scale from 0 to 4 and iris hyperemia on a scale of 0 to 2, whereas conjunctival congestion, swelling, and discharge were graded on the following scales of 0– 3, 0–4, and 0–3, respectively. The total mean scores for each treatment group were calculated. The evaluation criteria considered the solutions as nonirritant for values ranging from 0 to 3.9, slightly irritant for values ranging from 4 to 8.9, moderately irritant for values from 9 to 12.9, and seriously irritant for values ranging from 13 to 16, respectively. 2.6. Statistical analysis
2.4. Evaluation of corneal hydration levels The corneal hydration level was evaluated using the gravimetric method by measuring the total water content of the cornea after desiccation (Monti et al., 2002). At the end of each permeation experiment, the cornea was removed from the perfusion apparatus. After the remaining sclera being carefully removed, the trimmed cornea was gently blotted dry and weighed. The cornea was dried overnight in an oven at 70 1C and weighed again. The percent corneal hydration level (HL%) was calculated by the following Eq. (4): HL% ¼ ð1W d =W w Þ 100
ð4Þ
where Wd and Ww are the dry corneal weight and wet corneal weight, respectively. 2.5. Ocular irritation tests Ocular irritation was evaluated according to the modified Draize test (Draize et al., 1944). A total of 24 rabbits were divided into four groups based on the various concentrations of borneol. Solutions consisting of 0, 0.05, 0.1 and 0.2% (w/v) borneol in phosphate buffer (PBS) at pH 7.4, along with 4% propylene glycol and 1% tween-80 were instilled into the left eyes, and blank PBS into the right eyes used as controls. A single instillation of the sample (100 ml) every 4 h and four times per day lasted for a period of 7 days. The ocular tissues were observed at 1, 12, 24, 48, and 72 h after the last instillation by slit-lamp biomicroscopy.
All the results are presented as mean7S.D. Statistical analysis was assessed by one-way analysis of variance (ANOVA) using SPSS 16.0.1 software (Spss Inc., Chicago, Illinois, USA). When ANOVA results showed statistically significant differences, post hoc testing was performed for intergroup comparisons using the least significant difference (LSD) test. Po0.05 was considered significant.
3. Results 3.1. In vitro transcorneal permeation studies The model drugs were divided into two types: lipophilic drugs and hydrophilic drugs. Rhodamine B is lipophilic drug, and sodium-fluorescein, FITC-dextrans of 4, 10, 20 and 40 kDa are hydrophilic drugs. The effect of borneol concentration on Papp value of the compound is shown in Fig. 2, which shows that the increasing tendencies of Papp values were in line with those of the concentration of borneol. The permeation parameters are summarized in Table 1. All regressions applied that each plot were significant (R2 40.9). As shown by the Papp and Jss values, 4% propylene glycol and 1% tween-80 as cosolvent did not increase corneal permeability compared with the control group (P40.05). As shown by the Papp values, permeability was enhanced significantly by 0.2% borneol for rhodamine B, sodium-fluorescein and FITC-dextrans of 4 kDa, but not for FITC-dextrans of 10, 20 and 40 kDa, in comparison with the control group. Very little leakage
Fig. 2. The corneal apparent permeability coefficients of drug solutions containing different concentrations of borneol (Each point represented as mean 7 S.D., n¼ 4.).
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Table 1 Effect of different concentrations of borneol upon the transcorneal permeation parameters of compounds across excised rabbit cornea. Compounds
Concentration (%, w/v)
Papp 106 (cm s 1)
Jss 103 (mg cm 2 s 1)
Lag time (min)
Enhancement ratio
Rhodamine B
0 Cosolvent 0.05 0.1 0.2 0 Cosolvent 0.05 0.1 0.2 0 Cosolvent 0.05 0.1 0.2 0 Cosolvent 0.05 0.1 0.2
6.196 7 0.667 6.430 7 0.552 9.254 7 0.541n 10.573 7 0.729n 11.260 7 0.888n 0.6907 0.301 0.7017 0.276 0.7017 0.073 1.334 7 0.304n 1.716 7 0.182n 0.2807 0.038 0.282 7 0.067 0.327 7 0.087 0.488 7 0.043 1.169 7 0.550n 0.0657 0.016 0.0677 0.012 0.0667 0.020 0.0687 0.013 0.0727 0.022
0.929 7 0.100 0.962 7 0.085 1.388 7 0.081n 1.586 7 0.109n 1.689 7 0.133n 0.102 7 0.047 0.105 7 0.041 0.105 7 0.022 0.2007 0.046n 0.258 7 0.027n 0.280 7 0.038 0.282 7 0.067 0.327 7 0.087 0.488 7 0.043 1.169 7 0.550n 0.065 7 0.016 0.067 7 0.012 0.066 7 0.020 0.068 7 0.013 0.072 7 0.022
59.31 75.60 60.69 77.99 72.15 78.74 67.12 711.62 70.93 79.08 75.67 724.14 65.13 725.48 66.15 75.09 64.96 724.09 51.39 723.63 58.83 715.28 56.31 74.99 57.81 716.97 75.19 715.56 58.28 77.54 67.46 730.28 79.48 725.34 70.70 724.82 81.89 727.68 58.97 79.54
1.00 1.04 1.49 1.71 1.82 1.00 1.02 1.02 1.93 2.49 1.00 1.01 1.17 1.74 4.18 1.00 1.03 1.02 1.05 1.11
Sodium-fluorescein
FD-4 kDa
FD-10 kDa
Each value represented as mean 7 S.D (n¼ 4). n Po0.05 vs control. Statistics of the data used one-way ANOVA followed by the least significant difference (LSD) test. Cosolvent: 4% propylene glycol and 1% tween-80. Papp—apparent permeability coefficients; Jss—steady-state flux.
Fig. 3. The correlation between molecular sizes and Papp of hydrophilic compounds with 0.2% borneol application (Each point represented as mean 7 S.D., n¼ 4.).
of FITC-dextrans of 20 and 40 kDa was observed and the permeability was extremely low (data for FITC-dextrans of 20 and 40 kDa was not shown). The enhancement ratio values for rhodamine B, sodium-fluorescein, FITC-dextrans of 4 and 10 kDa were 1.82 (Po0.05), 2.49 (Po0.05), 4.18 (Po0.05) and 1.11 (not significant), respectively. The increasing and decreasing tendencies of Jss values were consistent with those of Papp values. Borneol did not significantly change the lag time, which represents the time needed for enhancement to reach maximum efficacy. The permeation results indicated that borneol could enhance Papp values for both hydrophilic and lipophilic compounds. We determined paracellular permeability (Papp) of cornea enhanced by 0.2% borneol using sodium-fluorescein and several FITC dextrans. These values are plotted in Fig. 3. It can be seen that the paracellular permeability enhanced by 0.2% borneol was correlate to the molecular weight of the model drugs (R2 ¼0.9976).
indicates a corneal damage (Monti et al., 2002). Table 2 presents the percent corneal hydration level values. All values were less than 83%, which indicated that borneol produced no damage to the isolated corneas during the studies. 3.3. Ocular irritation tests Ocular irritation tests were carried out to examine the effect of borneol on the corneal structure and integrity (Draize et al., 1944). The average score was 0, 0, 0.6770.52, and 1.5070.55 at concentrations of 0%, 0.05%, 0.1%, and 0.2% (w/v) borneol, respectively. In none of the six rabbits did the score exceed 4. In addition, no visible ocular damage or abnormal clinical signs were observed in the cornea, iris or conjunctiva on administering borneol solutions of different concentration. These results indicated that the enhancer was nonirritant and could be tolerated by rabbit eyes.
3.2. Evaluation of corneal hydration levels 4. Discussion The corneal tissue hydration levels are considered as a sensitive indicator of corneal damage in vitro. Generally, the normal hydration level ranges from 76% to 80%. A value over 83%
Borneol is frequently used in the preparation of topical analgesic, antipruritic, and anti-inflammatory drugs, and as a
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Table 2 Values of rabbit corneal hydration affected by model compounds with different concentrations of borneol. Concentration (%, w/v)
0 Cosolvent 0.05 0.1 0.2
Corneal hydration levels (%) Sodiumfluorescein
Rhodamine B
FD-4 kDa
FD-10 kDa
78.95 7 1.23 78.62 7 1.89 78.75 7 1.24 78.94 7 2.18 80.31 7 1.46
77.69 7 2.42 76.52 7 5.34 78.90 7 1.81 76.12 7 2.84 76.97 7 3.66
77.58 7 1.98 77.57 7 1.54 77.84 7 4.60 77.907 5.21 76.94 7 3.95
78.57 71.85 77.56 74.33 77.04 74.45 78.52 71.31 77.48 73.93
Each value represented as mean 7 S.D (n¼ 4). Cosolvent: 4% propylene glycol and 1% tween-80.
penetration enhancer of drugs (Mai et al., 2003). Borneol is also used in some ophthalmic preparation for relieving visual fatigue and eye discomfort, such as Pearl Eye Drop (Guilin Jiqi Medicion Company, Guilin, China) which was first used in 2002 (Li et al., 2010). But borneol is mainly used as an assistant in prescription. Some previous studies reported the ability of borneol in improving the drug delivery into the eye tissues (Li et al., 2010; Yang et al., 2009; Wu et al., 2006). In this study, we employed isolated rabbit cornea to test the hypothesis that borneol could enhance transcorneal permeability of compounds with different hydrophilicities and molecular sizes in vitro. The results showed that borneol can enhance rhodamine B, sodium-fluorescein and FITCdextrans of 4 kDa into the eye through the transcorneal pathways without causing ocular irritation and toxicity. However, borneol failed to increase significantly corneal permeation of FITCdextrans of 10, 20, and 40 kDa. The corneal membrane consists of three essential layers, epithelium, stroma, and endothelium. The epithelium and stroma are the main barriers to drug absorption. The epithelium is a rate-limiting membrane for hydrophilic compounds, whereas the stroma is a rate-limiting membrane for lipophilic compounds, which permeated the excised cornea more rapidly (Robinson, 1993; Salama et al., 2006). Permeation of highly lipophilic compounds across celling membrane takes place via transcellular routes and hydrophilic compounds across the tight connection between cells use paracellular route (Afouna et al., 2010). Yang et al. (2009) showed borneol could promote the corneal permeability of two lipophilic and three hydrophilic drugs in vitro, especially hydrophilic drugs. Our study showed 0.2% borneol can enhance both lipophilic and hydrophilic compounds through the cornea. We also found that the enhancement ratios of hydrophilic compounds (sodium-fluorescein, enhancement ratio ¼2.49, and FITC-dextrans of 4 kDa, enhancement ratio ¼4.18) were more than the enhancement ratio of lipophilic compound (rhodamine B, enhancement ratio ¼1.82). On the basis of enhancement ratios, we concluded that the increased transcorneal permeation was more significant for hydrophilic compounds than lipophilic ones. Above results are consistent with Yang’s results. As an enhancer, the concentration of borneol also plays an important role on the apparent permeability coefficient. Compared with Wu et al. (2006) and Yang et al. (2009) only investigated one borneol concentration (0.1%), we investigated the enhancing permeation effect of 0.05%, 0.1% and 0.2% borneol in the present study. We found that the permeability coefficient of model compounds increases with the concentration of borneol increased, indicating that borneol enhanced the corneal penetration of model compounds in a concentration-dependent manner. In addition, different lag times suggested that borneol or model compounds affect the stroma and endothelium differently. Further study is necessary to investigate the mechanism of these ingredients in the corneal composite structure.
The mechanism of transcorneal permeation enhancement of penetration enhancers might include increasing the permeability of cell membranes or loosening the tight junctions or both (Sultana et al., 2006). As detected in the previous experiment, borneol can reversibly rearrange the sequence of the phospholipids from the lipid bilayer of the corneal epithelium, make it more regular, and thus, increasing permeation of surface epithelium cells (Fan et al., 1998). It is quite possible that borneol facilitates the transcorneal passage of drugs by reducing drag of phospholipids of the lipid bilayer in the corneal epithelium, which is the main barrier for topically applied drugs. Jin et al. (2011) showed that borneol could regulate the reversible translocation of two tight junction proteins (claudin-5 and occluding) between the cell membrane and the cytoplasm to improve the permeability of the blood–optic nerve barrier, while the borneol did not change the levels of claudin-5 or occluding. Our results suggested that the in vitro increase in permeability across cornea depends on the dosage of borneol. Moreover, there was no substantial damage to the cornea because hydration level was quite normal. It is also quite possible that borneol loosens the corneal epithelium junctions reversibly when present in high concentrations in the epithelium. Above mechanisms may give reasons for why borneol can enhance both lipophilic and hydrophilic compounds. In addition, our results showed that the increased transcorneal permeation was more significant for hydrophilic compounds than lipophilic ones. This is possibly due to the formation of a hydration barrier or the inherent preferential permeation of hydrophilic compounds by the corneal stroma (Liu et al., 2011). Besides lipophilicity and hydrophilicity of drugs, permeation of drugs across the cornea also depends on other drug properties such as solubility, molecular size and shape, charge and degree of ionization. Our studies investigate correlation between molecular sizes and Papp of hydrophilic compounds with 0.2% borneol application. The permeability coefficient of model compounds decreases with the molecular weight of hydrophilic compounds increased. Papp was a linear function of the molecular weight, which is consistent with all labels traversing the same diffusional pathway. Moreover, 0.2% borneol merely enhanced transcorneal permeation of hydrophilic compounds with molecular weight 4 kDa or less. The effective diameter of 40 kDa dextran has been ˚ which is consistent with priorly determined reported to be 46 A, corneal endothelial junctional width values of 38–43 A˚ (Ma et al., 2007; Fischbarg et al., 1977). The effective hydrodynamic radius of 4 kDa dextran has been reported to be 13 A˚ (Ambati et al., 2000). The present results indicated that borneol quite possibly loosens the epithelium junctions in cornea and the extent is within a limit. The cornea is highly sensitive to irritants, with resulting eye blink, lacrimation, inflammation and even vision loss. The corneal epithelium and endothelium restrict water transfer to the stroma. Epithelial or endothelial damage allows extra water enter the stroma leading to corneal edema. The hydration value of the cornea is an important index for evaluating tissue irritability in vitro. The normal hydration value of the cornea is between 76% and 80%. A hydration value increasing to 83% or above infers that the cornea has suffered from certain damage (Monti et al., 2002). We studied the corneal hydration level and irritation after administering different concentrations of borneol solutions. None of the percent corneal hydration level values were greater than 83%, indicating that the borneol did not cause any substantial damage to the epithelium or endothelium. Ocular irritation tests indicated a good corneal biocompatibility of borneol. In general, borneol exhibited a fair ocular tolerability at broad concentrations of 0.05%–0.2% (w/v). In previous studies of our lab, our colleagues Yang et al. (2009), Li et al. (2010), Liu et al. (2012), Jin et al. (2011) and Xu et al. (2011) have assessed the short-term and long-term
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safety of borneol on the basis of corneal hydration level, Draize test, electrophysiological, and histological examination. No substantial toxic reactions were observed in corneal hydration level, electrophysiological, or histological examinations after adding the borneol in our colleagues’ studies. Therefore, it can be concluded that the use of borneol is quite safe in the administration of topical drop. In addition, borneol has been widely used for ocular disease in China and the clinical practice has proved the long term clinical safety of low dose borneol at some extent. However, it is necessary to conduct further safety studies before using it clinically. In conclusion, this study evaluated borneol as an ocular drug delivery enhancer for the compounds with different hydrophilicities and molecular sizes. We demonstrated that borneol could enhance transcorneal permeation of both hydrophilic and lipophilic compounds, especially hydrophilic ones. Furthermore, 0.2% borneol can enhance transcorneal permeation of hydrophilic compounds with molecular weight r4 kDa. Additionally, it was nonirritant even at relatively high concentrations. Therefore, borneol can be considered as a safe and effective penetration enhancer for ocular drug administration. Our study indicates the role of borneol, and states that it might be possible to be applied as an adjuvant during the eye treatments. Further studies are needed to confirm the results and to explore the performance of borneol in ophthalmic drug delivery system. References Afouna, M.I., Khedr, A., Abdel-Naim, A.B., Al-Marzoqi, A., 2010. Influence of various concentrations of terpene-4-ol enhancer and carbopol-934 mucoadhesive upon the in vitro ocular transport and the in vivo intraocular pressure lowering effects of dorzolamide ophthalmic formulations using albino rabbits. J. Pharm. Sci. 99, 119–127. Ambati, J., Canakis, C., Miller, J.W., Gragoudas, E., Edwards, A., Weissgold, D.J., Kim, I., Delori, F., Adamis, A.P., 2000. Diffusion of high molecular weight compounds through sclera. Invest. Ophthalmol. Vis. Sci. 41, 1181–1185. Bucolo, C., Drago, F., Salomone, S., 2012. Ocular drug delivery: a clue from nanotechnology. Front. Pharmacol. 3, 188, http://dx.doi.org/10.3389/fphar.2012.00188. Cai, Z., Hou, S., Li, Y., Zhao, B., Yang, Z., Xu, S., Pu, J., 2008. Effect of borneol on the distribution of gastrodin to the brain in mice via oral administration. J. Drug Target. 16, 178–184. Cui, Y., Li, L., Zhang, L., Li, J., Gu, J., Gong, H., Guo, P., Tong, W., 2011. Enhancement and mechanism of transdermal absorption of terpene-induced propranolol hydrochloride. Arch. Pharm. Res. 34, 1477–1485. Davies, N.M., 2000. Biopharmaceutical considerations in topical ocular drug delivery. Clin. Exp. Pharmacol. Physiol. 27, 558–562. Draize, J.H., Woodard, G., Calvery, H.O., 1944. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 82, 377–390.
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Eljarrat-Binstock, E., Pe’er, J., Domb, A.J., 2010. New techniques for drug delivery to the posterior eye segment. Pharm. Res. 27, 530–543. Fan, L., Tan, Y., Lu, J., Lu, L., Wang, L., 1998. An experimental research on osmosis of cytomembrane of corneal epithelium promoted by Borneolum in the rabbits eye. J. Trad. Chin. Ophthalmol. 8, 67–69. (Article in Chinese). Fischbarg, J., Warshavsky, C.R., Lim, J.J., 1977. Pathways for hydraulically and osmotically-induced water flows across epithelia. Nature 266, 71–74. Jin, D., Wang, F., Qu, L., Li, Z., Jin, L., Liu, P., Xu, X., Cui, H., 2011. The distribution and expression of claudin-5 and occluding at the rat blood–optic nerve barrier after borneol treatment. Mol. Biol. Rep. 38, 913–920. Kaur, I.P., Smitha, R., 2002. Penetration enhancers and ocular bioadhesives: two new avenues for ophthalmic drug delivery. Drug. Dev. Ind. Pharm. 28, 353–369. Li, Z., Sun, D., Yang, H., Liu, X., Luan, L., Bai, J., Cui, H., 2010. Effect of borneol on the distribution of danshensu to the eye in rabbit via oral administration. Curr. Eye Res. 35, 565–572. Liu, J., Fu, S., Wei, N., Hou, Y., Zhang, X., Cui, H., 2012. The effects of combined menthol and borneol on fluconazole permeation through the cornea ex vivo. Eur. J. Pharmacol. 5 (688), 1–5. Liu, R., Liu, Z., Zhang, C., Zhang, B., 2011. Gelucire44/14 as a novel absorption enhancer for drugs with different hydrophilicities: in vitro and in vivo improvement on transcorneal permeation. J. Pharm. Sci. 100, 3186–3195. Lu, Y., Du, S., Yao, Z., Zhao, P., Zhai, Y., 2009. Study on natural borneol and synthetic borneol affecting mucosal permeability of gardenia extract. Zhongguo Zhong Yao Za Zhi 34, 1207–1210. (Article in Chinese). Ma, L., Kuang, K., Smith, R.W., Rittenband, D., Iserovich, P., Diecke, F.P.J, Fischbarg, J., 2007. Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium. Exp. Eye Res. 84, 790–798. Mai, L.M., Lin, C.Y., Chen, C.Y., Tsai, Y.C., 2003. Synergistic effect of bismuth subgallate and borneol, the major components of Sulbogin, on the healing of skin wound. Biomaterials 24, 3005–3012. Monti, D., Chetoni, P., Burgalassi, S., Najarro, M., Saettone, M.F., 2002. Increased corneal hydration induced by potential ocular penetration enhancers: assessment bydifferential scanning calorimetry (DSC) and by desiccation. Int. J. Pharm. 232, 139–147. Rawas-Qalaji, M., Williams, C.A., 2012. Advances in ocular drug delivery. Curr. Eye Res. 37, 345–356. Robinson, J.R., 1993. Ocular anatomy and physiology relevant to ocular drug delivery. In: Mitra, A.K. (Ed.), Ophthalmic Drug Delivery Systems. Marcel Dekker, New York, pp. 29–57. Sahoo, S.K., Dilnawaz, F., Krishnakuma, S., 2008. Nanotechnology in ocular drug delivery. Drug Discovery Today 13, 144–151. Salama, N.N., Eddington, N.D., Fasano, A., 2006. Tight junction modulation and its relationship to drug delivery. Adv. Drug Deliv. Rev. 58, 15–28. Sultana, Y., Jain, R., Aqil, M., Ali, A., 2006. Review of ocular drug delivery. Curr. Drug Deliv. 3, 207–217. Wu, C.J., Huang, Q.W., Qi, H.Y., Guo, P., Hou, S.X., 2006. Promoting effect of borneol on the permeability of puerarin eye drops and timolol maleate eye drops through the cornea in vitro. Pharmazie 61, 783–788. Xu, X., Yu, N., Bai, Z., Xun, Y., Jin, D., Li, Z., Cui, H., 2011. Effect of menthol on ocular drug delivery. Graefe’s Arch. Clin. Exp. Ophthalmol. 249, 1503–1510. Yang, H., Xun, Y., Li, Z., Hang, T., Zhang, X., Cui, H., 2009. Influence of borneol on in vitro corneal permeability and on in vivo and in vitro corneal toxicity. J. Int. Med. Res. 37, 791–802.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
In vitro evaluation of enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes Hui-Ping Qi a,f,1, Xiang-Chun Gao b,1, Li-Qiong Zhang a, Shu-Qin Wei c, Sheng Bi d,e, Zi-Chao Yang f, Hao Cui a,n a
Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, China Department of Ophthalmology, Harbin Medical University, Harbin, China Perinatal Epidemiology, CHU Sainte-Justine University of Montreal, Montreal, Canada d Department of Neurology, the First Affiliated Hospital of Harbin Medical University, China e Central Laboratory, the First Affiliated Hospital of Harbin Medical University, China f Department of Neurology, the Fourth Affiliated Hospital of Harbin Medical University, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 August 2012 Received in revised form 8 February 2013 Accepted 14 February 2013 Available online 28 February 2013
To investigate the enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes. Six compounds, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa were selected as model drugs. Permeation studies were performed using excised cornea of rabbits by a Franz-type diffusion apparatus. The safety of borneol was assessed on the basis of corneal hydration level and Draize eye test. The application of 0.2% borneol to the cornea increased the apparent permeability coefficient by 1.82—(P o 0.05), 2.49—(P o0.05), 4.18—(P o 0.05) and 1.11-fold (not significant) for rhodamine B, sodium-fluorescein, FITC-dextrans of 4 and 10 kDa, respectively. No significant permeability enhancement of FITC dextrans of 10, 20 and 40 kDa with borneol was found compared to control. The permeability coefficient enhanced by 0.2% borneol was linear correlated to the molecular weight of model drugs (R2 ¼ 0.9976). With the 0.05%, 0.1% and 0.2% borneol application, the corneal hydration values were o83% and Draize scores were o 4. Borneol may improve the transcorneal penetration of both hydrophilic and lipophilic compounds without causing toxic reactions, especially hydrophilic ones. Furthermore, 0.2% borneol can enhance the permeation of hydrophilic compounds with molecular weight r 4 kDa. Hence, borneol can be considered as a safe and effective penetration enhancer for ocular drug administration. & 2013 Elsevier B.V. All rights reserved.
Keywords: Permeability enhancers Borneol Transcorneal permeation Ocular irritation
1. Introduction Most ophthalmic drugs are administrated topically in the form of eye drops due to its convenience and safety (Eljarrat-Binstock et al., 2010; Rawas-Qalaji and Williams, 2012). The cornea consisted of three primary layers, the epithelium, stroma and endothelium, is generally recognized as the major route of ocular penetration for topically instilled drugs (Robinson, 1993). The lipophilic corneal epithelium contains 5–7 layers of cells each connected by tight junctions and restricts the diffusion of most hydrophilic drugs through the paracellular pathway; while the stroma, which is mainly composed of hydrated collagen, exerts a
n Correspondence to: Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, 23rd Youzheng Street, Nangang District, Harbin, Heilongjiang Province 150001, China. E-mail address: [email protected] (H. Cui). 1 Hui-Ping Qi and Xiang-Chun Gao contributed equally to this work.
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.02.031
diffusional barrier to highly lipophilic drugs through the transcellular pathway (Robinson, 1993; Salama et al., 2006; Bucolo et al., 2012). Though the endothelium is not a significant barrier to the transcorneal diffusion, its permeability depends on molecular weight rather than characteristic of compound (Bucolo et al., 2012). Therefore, it is quite difficult to ensure a successful delivery of drugs to the intended area in the eye through local application. The ocular bioavailability is very low, typically less than 5% (Eljarrat-Binstock et al., 2010; Sahoo et al., 2008; Davies, 2000). Various attempts have been made to improve drug bioavailability by increasing corneal drug penetration. An alternative approach is to transiently increase the corneal permeation with adding appropriate penetration enhancers to drug formulas. An ideal penetration enhancer is nontoxic, nonirritant, chemically and pharmacologically inert, and compatible with formulation ingredients (Liu et al., 2011). In addition, it should have a fast and reversible onset of action and should be effective at low concentrations (Liu et al., 2011). So far, many enhancers, such as
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fluorescein and rhodamine B were obtained from Melone Pharmaceutical Co., Ltd. (Dalian, China). Other chemicals were of reagent grade and obtained from Hengxing Pure Chemical Co., Ltd. (Tianjin, China). 2.2. Animals
Fig. 1. Chemical structure of borneol.
benzalkonium chloride, ethylenediamine tetra-acetic acid (EDTA), nonionic surfactants, surface-active heteroglycosides, bile salts, polycarbophil–cysteine conjugates and boric acid have been used (Kaur and Smitha, 2002). However, some of them cause significant ocular irritation or toxicity even at low concentrations. Borneol (C10H18O, molecular weight, 154.24) is a naturallyoccurring cyclic terpene alcohol extracted from the resin and volatile oil of dipterocarp (Fig. 1). It has been extensively used in pharmaceutical preparation of ophthalmic ointment, topical drops and Chinese herbal drugs in China and other Asian countries (Li et al., 2010). Some studies showed that borneol can act as a penetration enhancer for a number of drugs administered through various physiologic barriers such as skin (Cui et al., 2011), brain (Cai et al., 2008), and mucous membrane (Lu et al., 2009). Recently, several studies indicated that borneol has the potential to be used as an ocular penetration enhancer (Li et al., 2010; Yang et al., 2009; Wu et al., 2006). Wu et al. (2006) reported that borneol enhanced permeation of puerarin through the isolated cornea but did not enhance the transcorneal permeation of maleate. Yang et al. (2009) showed that borneol could promote the corneal permeability of two lipophilic (indometacin and dexamethasone) and three hydrophilic drugs (ofloxacin, tobramycin and virazle) in vitro. Li et al. (2010) showed that borneol could enhance the diffusion of danshensu across the ocular–blood barrier. Jin et al. (2011) also reported that it can reversibly open blood–optic nerve barrier without causing any damage to the blood–optic nerve barrier. As we all know, patient compliance and comfort considerations in drug administration may impact the drug’s therapeutic efficacy. Compared to other penetration enhancers, the major advantages of borneol are the wide use for ocular disease and the long term clinical safety. Currently, studies reported that borneol could enhance corneal permeability for drugs with low molecular weight. More compounds with different molecular weights, especially high molecular weight, were not tested. Further studies are still essential to verify the penetration-enhancing effect of borneol on ocular absorption and to explore the relevant mechanisms. The present study was designed to explore the transcorneal permeation enhancing effect of borneol in vitro on a variety of compounds with different hydrophilicities and molecular sizes, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa. Potential toxicity produced by borneol was assessed in vitro by measuring corneal hydration levels and in vivo by Draize eye test.
2. Materials and methods 2.1. Materials Borneol, FITC dextrans of 4, 10, 20 and 40 kDa were obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, U.S.A.). Sodium-
New Zealand white rabbits weighing 2.5–3.0 kg were provided by Institute of Experimental Animals, Harbin Medical University. The animals were given food and water ad libitum. The animal room was well ventilated, and a regular 12 h:12 h light–dark cycle was maintained throughout the experimental period. Animal care and protocols were in accordance with institutional guidelines and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Research. 2.3. In vitro transcorneal permeation studies The transcorneal permeation studies were evaluated across excised rabbit corneas. In brief, the rabbits were sacrificed by injecting air into the marginal ear vein. The cornea was dissected with a scleral ring (5 mm laterally from the limbus), and the lens and iris were carefully removed. Freshly excised rabbit corneas were immediately mounted over the modified Franz-type vertical diffusion chambers. Blank glutathione bicarbonate ringer (GBR) buffer (9 ml, pH 7.4) was added to the endothelial side and 1 ml of donor solution of the compounds with various concentrations of borneol (0%, 0.05%, 0.1%, and 0.2%, w/v) was added to the epithelial side. Borneol was dissolved in the GBR buffer containing 4% propylene glycol and 1% tween-80 as cosolvent. Sodiumfluorescein (molecular weight, 376) and rhodamine B (molecular weight, 479) were both at a concentration of 150 mg/ml, and FITCdextrans of 4, 10, 20 and 40 kDa were at a concentration of 1 mg/ ml. The solutions in each chamber were mixed by bubbling an O2–CO2 (95:5) mixture at the rate of three to four bubbles per second and maintaining the temperature at 35 71 1C under mixing conditions using a constant magnetic stirring with a rotating speed of 600 rpm. The corneal area available for diffusion was 0.785 cm2. At intervals of 30 min, until 240 min, samples of 200 ml were taken from the endothelial side and replaced by an equal volume of blank GBR buffer. Concentrations of sodiumfluorescein, FITC-dextrans of 4, 10, 20 and 40 kDa were determined using a multi-mode microplate reader (SpectraMax M5, Molecular Devices, USA) with 485-nm excitation and 530-nm emission filters. Concentrations of rhodamine B were determined by the multi-mode microplate reader with 530-nm excitation and 590-nm emission filters. All the experiments were conducted in four repeats. The corneal permeation parameters were calculated from the plot of cumulative amount of compounds permeated as a function of time. The accumulated amounts of compounds permeated across the cornea were calculated by the following Eq. (1): ! n1 n1 X V X Q ¼ V 0 Cn þ Ci ¼ V 0Cn þ V Ci ð1Þ V0 i ¼ 1 i¼1 Q—the accumulated permeation amount within time t, Cn—the measured concentration value within time t, V0—the total volume of solution in the reservoir chamber, V—the sampling volume per time point, Ci—the measured value of concentration before time t. The apparent permeability coefficient (Papp) and steady-state flux (Jss) of the compounds were calculated by the following Eqs. (2) and (3), assuming passive diffusion under steady-state conditions: Papp ¼
DQ Dt C 0 A 60
ð2Þ
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J ss ¼ C 0 P app
ð3Þ
where DQ/Dt is the steady-state slope of the linear portion of the cumulative permeated amount of drug in the receiving chamber (Q) versus time (t), C0 is the initial concentration of the compound dissolved in the epithelial donor chamber, A is the exposed corneal surface area (0.785 cm2), and 60 is the factor for conversion of minute to second. The lag time calculated for each plot was the time axis intercept of the regression line. The Papp increase was measured by the enhancement ratio, calculated as the ratio between the Papp values obtained in the absence and presence of borneol.
The corneal opacity was graded on a scale from 0 to 4 and iris hyperemia on a scale of 0 to 2, whereas conjunctival congestion, swelling, and discharge were graded on the following scales of 0– 3, 0–4, and 0–3, respectively. The total mean scores for each treatment group were calculated. The evaluation criteria considered the solutions as nonirritant for values ranging from 0 to 3.9, slightly irritant for values ranging from 4 to 8.9, moderately irritant for values from 9 to 12.9, and seriously irritant for values ranging from 13 to 16, respectively. 2.6. Statistical analysis
2.4. Evaluation of corneal hydration levels The corneal hydration level was evaluated using the gravimetric method by measuring the total water content of the cornea after desiccation (Monti et al., 2002). At the end of each permeation experiment, the cornea was removed from the perfusion apparatus. After the remaining sclera being carefully removed, the trimmed cornea was gently blotted dry and weighed. The cornea was dried overnight in an oven at 70 1C and weighed again. The percent corneal hydration level (HL%) was calculated by the following Eq. (4): HL% ¼ ð1W d =W w Þ 100
ð4Þ
where Wd and Ww are the dry corneal weight and wet corneal weight, respectively. 2.5. Ocular irritation tests Ocular irritation was evaluated according to the modified Draize test (Draize et al., 1944). A total of 24 rabbits were divided into four groups based on the various concentrations of borneol. Solutions consisting of 0, 0.05, 0.1 and 0.2% (w/v) borneol in phosphate buffer (PBS) at pH 7.4, along with 4% propylene glycol and 1% tween-80 were instilled into the left eyes, and blank PBS into the right eyes used as controls. A single instillation of the sample (100 ml) every 4 h and four times per day lasted for a period of 7 days. The ocular tissues were observed at 1, 12, 24, 48, and 72 h after the last instillation by slit-lamp biomicroscopy.
All the results are presented as mean7S.D. Statistical analysis was assessed by one-way analysis of variance (ANOVA) using SPSS 16.0.1 software (Spss Inc., Chicago, Illinois, USA). When ANOVA results showed statistically significant differences, post hoc testing was performed for intergroup comparisons using the least significant difference (LSD) test. Po0.05 was considered significant.
3. Results 3.1. In vitro transcorneal permeation studies The model drugs were divided into two types: lipophilic drugs and hydrophilic drugs. Rhodamine B is lipophilic drug, and sodium-fluorescein, FITC-dextrans of 4, 10, 20 and 40 kDa are hydrophilic drugs. The effect of borneol concentration on Papp value of the compound is shown in Fig. 2, which shows that the increasing tendencies of Papp values were in line with those of the concentration of borneol. The permeation parameters are summarized in Table 1. All regressions applied that each plot were significant (R2 40.9). As shown by the Papp and Jss values, 4% propylene glycol and 1% tween-80 as cosolvent did not increase corneal permeability compared with the control group (P40.05). As shown by the Papp values, permeability was enhanced significantly by 0.2% borneol for rhodamine B, sodium-fluorescein and FITC-dextrans of 4 kDa, but not for FITC-dextrans of 10, 20 and 40 kDa, in comparison with the control group. Very little leakage
Fig. 2. The corneal apparent permeability coefficients of drug solutions containing different concentrations of borneol (Each point represented as mean 7 S.D., n¼ 4.).
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Table 1 Effect of different concentrations of borneol upon the transcorneal permeation parameters of compounds across excised rabbit cornea. Compounds
Concentration (%, w/v)
Papp 106 (cm s 1)
Jss 103 (mg cm 2 s 1)
Lag time (min)
Enhancement ratio
Rhodamine B
0 Cosolvent 0.05 0.1 0.2 0 Cosolvent 0.05 0.1 0.2 0 Cosolvent 0.05 0.1 0.2 0 Cosolvent 0.05 0.1 0.2
6.196 7 0.667 6.430 7 0.552 9.254 7 0.541n 10.573 7 0.729n 11.260 7 0.888n 0.6907 0.301 0.7017 0.276 0.7017 0.073 1.334 7 0.304n 1.716 7 0.182n 0.2807 0.038 0.282 7 0.067 0.327 7 0.087 0.488 7 0.043 1.169 7 0.550n 0.0657 0.016 0.0677 0.012 0.0667 0.020 0.0687 0.013 0.0727 0.022
0.929 7 0.100 0.962 7 0.085 1.388 7 0.081n 1.586 7 0.109n 1.689 7 0.133n 0.102 7 0.047 0.105 7 0.041 0.105 7 0.022 0.2007 0.046n 0.258 7 0.027n 0.280 7 0.038 0.282 7 0.067 0.327 7 0.087 0.488 7 0.043 1.169 7 0.550n 0.065 7 0.016 0.067 7 0.012 0.066 7 0.020 0.068 7 0.013 0.072 7 0.022
59.31 75.60 60.69 77.99 72.15 78.74 67.12 711.62 70.93 79.08 75.67 724.14 65.13 725.48 66.15 75.09 64.96 724.09 51.39 723.63 58.83 715.28 56.31 74.99 57.81 716.97 75.19 715.56 58.28 77.54 67.46 730.28 79.48 725.34 70.70 724.82 81.89 727.68 58.97 79.54
1.00 1.04 1.49 1.71 1.82 1.00 1.02 1.02 1.93 2.49 1.00 1.01 1.17 1.74 4.18 1.00 1.03 1.02 1.05 1.11
Sodium-fluorescein
FD-4 kDa
FD-10 kDa
Each value represented as mean 7 S.D (n¼ 4). n Po0.05 vs control. Statistics of the data used one-way ANOVA followed by the least significant difference (LSD) test. Cosolvent: 4% propylene glycol and 1% tween-80. Papp—apparent permeability coefficients; Jss—steady-state flux.
Fig. 3. The correlation between molecular sizes and Papp of hydrophilic compounds with 0.2% borneol application (Each point represented as mean 7 S.D., n¼ 4.).
of FITC-dextrans of 20 and 40 kDa was observed and the permeability was extremely low (data for FITC-dextrans of 20 and 40 kDa was not shown). The enhancement ratio values for rhodamine B, sodium-fluorescein, FITC-dextrans of 4 and 10 kDa were 1.82 (Po0.05), 2.49 (Po0.05), 4.18 (Po0.05) and 1.11 (not significant), respectively. The increasing and decreasing tendencies of Jss values were consistent with those of Papp values. Borneol did not significantly change the lag time, which represents the time needed for enhancement to reach maximum efficacy. The permeation results indicated that borneol could enhance Papp values for both hydrophilic and lipophilic compounds. We determined paracellular permeability (Papp) of cornea enhanced by 0.2% borneol using sodium-fluorescein and several FITC dextrans. These values are plotted in Fig. 3. It can be seen that the paracellular permeability enhanced by 0.2% borneol was correlate to the molecular weight of the model drugs (R2 ¼0.9976).
indicates a corneal damage (Monti et al., 2002). Table 2 presents the percent corneal hydration level values. All values were less than 83%, which indicated that borneol produced no damage to the isolated corneas during the studies. 3.3. Ocular irritation tests Ocular irritation tests were carried out to examine the effect of borneol on the corneal structure and integrity (Draize et al., 1944). The average score was 0, 0, 0.6770.52, and 1.5070.55 at concentrations of 0%, 0.05%, 0.1%, and 0.2% (w/v) borneol, respectively. In none of the six rabbits did the score exceed 4. In addition, no visible ocular damage or abnormal clinical signs were observed in the cornea, iris or conjunctiva on administering borneol solutions of different concentration. These results indicated that the enhancer was nonirritant and could be tolerated by rabbit eyes.
3.2. Evaluation of corneal hydration levels 4. Discussion The corneal tissue hydration levels are considered as a sensitive indicator of corneal damage in vitro. Generally, the normal hydration level ranges from 76% to 80%. A value over 83%
Borneol is frequently used in the preparation of topical analgesic, antipruritic, and anti-inflammatory drugs, and as a
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Table 2 Values of rabbit corneal hydration affected by model compounds with different concentrations of borneol. Concentration (%, w/v)
0 Cosolvent 0.05 0.1 0.2
Corneal hydration levels (%) Sodiumfluorescein
Rhodamine B
FD-4 kDa
FD-10 kDa
78.95 7 1.23 78.62 7 1.89 78.75 7 1.24 78.94 7 2.18 80.31 7 1.46
77.69 7 2.42 76.52 7 5.34 78.90 7 1.81 76.12 7 2.84 76.97 7 3.66
77.58 7 1.98 77.57 7 1.54 77.84 7 4.60 77.907 5.21 76.94 7 3.95
78.57 71.85 77.56 74.33 77.04 74.45 78.52 71.31 77.48 73.93
Each value represented as mean 7 S.D (n¼ 4). Cosolvent: 4% propylene glycol and 1% tween-80.
penetration enhancer of drugs (Mai et al., 2003). Borneol is also used in some ophthalmic preparation for relieving visual fatigue and eye discomfort, such as Pearl Eye Drop (Guilin Jiqi Medicion Company, Guilin, China) which was first used in 2002 (Li et al., 2010). But borneol is mainly used as an assistant in prescription. Some previous studies reported the ability of borneol in improving the drug delivery into the eye tissues (Li et al., 2010; Yang et al., 2009; Wu et al., 2006). In this study, we employed isolated rabbit cornea to test the hypothesis that borneol could enhance transcorneal permeability of compounds with different hydrophilicities and molecular sizes in vitro. The results showed that borneol can enhance rhodamine B, sodium-fluorescein and FITCdextrans of 4 kDa into the eye through the transcorneal pathways without causing ocular irritation and toxicity. However, borneol failed to increase significantly corneal permeation of FITCdextrans of 10, 20, and 40 kDa. The corneal membrane consists of three essential layers, epithelium, stroma, and endothelium. The epithelium and stroma are the main barriers to drug absorption. The epithelium is a rate-limiting membrane for hydrophilic compounds, whereas the stroma is a rate-limiting membrane for lipophilic compounds, which permeated the excised cornea more rapidly (Robinson, 1993; Salama et al., 2006). Permeation of highly lipophilic compounds across celling membrane takes place via transcellular routes and hydrophilic compounds across the tight connection between cells use paracellular route (Afouna et al., 2010). Yang et al. (2009) showed borneol could promote the corneal permeability of two lipophilic and three hydrophilic drugs in vitro, especially hydrophilic drugs. Our study showed 0.2% borneol can enhance both lipophilic and hydrophilic compounds through the cornea. We also found that the enhancement ratios of hydrophilic compounds (sodium-fluorescein, enhancement ratio ¼2.49, and FITC-dextrans of 4 kDa, enhancement ratio ¼4.18) were more than the enhancement ratio of lipophilic compound (rhodamine B, enhancement ratio ¼1.82). On the basis of enhancement ratios, we concluded that the increased transcorneal permeation was more significant for hydrophilic compounds than lipophilic ones. Above results are consistent with Yang’s results. As an enhancer, the concentration of borneol also plays an important role on the apparent permeability coefficient. Compared with Wu et al. (2006) and Yang et al. (2009) only investigated one borneol concentration (0.1%), we investigated the enhancing permeation effect of 0.05%, 0.1% and 0.2% borneol in the present study. We found that the permeability coefficient of model compounds increases with the concentration of borneol increased, indicating that borneol enhanced the corneal penetration of model compounds in a concentration-dependent manner. In addition, different lag times suggested that borneol or model compounds affect the stroma and endothelium differently. Further study is necessary to investigate the mechanism of these ingredients in the corneal composite structure.
The mechanism of transcorneal permeation enhancement of penetration enhancers might include increasing the permeability of cell membranes or loosening the tight junctions or both (Sultana et al., 2006). As detected in the previous experiment, borneol can reversibly rearrange the sequence of the phospholipids from the lipid bilayer of the corneal epithelium, make it more regular, and thus, increasing permeation of surface epithelium cells (Fan et al., 1998). It is quite possible that borneol facilitates the transcorneal passage of drugs by reducing drag of phospholipids of the lipid bilayer in the corneal epithelium, which is the main barrier for topically applied drugs. Jin et al. (2011) showed that borneol could regulate the reversible translocation of two tight junction proteins (claudin-5 and occluding) between the cell membrane and the cytoplasm to improve the permeability of the blood–optic nerve barrier, while the borneol did not change the levels of claudin-5 or occluding. Our results suggested that the in vitro increase in permeability across cornea depends on the dosage of borneol. Moreover, there was no substantial damage to the cornea because hydration level was quite normal. It is also quite possible that borneol loosens the corneal epithelium junctions reversibly when present in high concentrations in the epithelium. Above mechanisms may give reasons for why borneol can enhance both lipophilic and hydrophilic compounds. In addition, our results showed that the increased transcorneal permeation was more significant for hydrophilic compounds than lipophilic ones. This is possibly due to the formation of a hydration barrier or the inherent preferential permeation of hydrophilic compounds by the corneal stroma (Liu et al., 2011). Besides lipophilicity and hydrophilicity of drugs, permeation of drugs across the cornea also depends on other drug properties such as solubility, molecular size and shape, charge and degree of ionization. Our studies investigate correlation between molecular sizes and Papp of hydrophilic compounds with 0.2% borneol application. The permeability coefficient of model compounds decreases with the molecular weight of hydrophilic compounds increased. Papp was a linear function of the molecular weight, which is consistent with all labels traversing the same diffusional pathway. Moreover, 0.2% borneol merely enhanced transcorneal permeation of hydrophilic compounds with molecular weight 4 kDa or less. The effective diameter of 40 kDa dextran has been ˚ which is consistent with priorly determined reported to be 46 A, corneal endothelial junctional width values of 38–43 A˚ (Ma et al., 2007; Fischbarg et al., 1977). The effective hydrodynamic radius of 4 kDa dextran has been reported to be 13 A˚ (Ambati et al., 2000). The present results indicated that borneol quite possibly loosens the epithelium junctions in cornea and the extent is within a limit. The cornea is highly sensitive to irritants, with resulting eye blink, lacrimation, inflammation and even vision loss. The corneal epithelium and endothelium restrict water transfer to the stroma. Epithelial or endothelial damage allows extra water enter the stroma leading to corneal edema. The hydration value of the cornea is an important index for evaluating tissue irritability in vitro. The normal hydration value of the cornea is between 76% and 80%. A hydration value increasing to 83% or above infers that the cornea has suffered from certain damage (Monti et al., 2002). We studied the corneal hydration level and irritation after administering different concentrations of borneol solutions. None of the percent corneal hydration level values were greater than 83%, indicating that the borneol did not cause any substantial damage to the epithelium or endothelium. Ocular irritation tests indicated a good corneal biocompatibility of borneol. In general, borneol exhibited a fair ocular tolerability at broad concentrations of 0.05%–0.2% (w/v). In previous studies of our lab, our colleagues Yang et al. (2009), Li et al. (2010), Liu et al. (2012), Jin et al. (2011) and Xu et al. (2011) have assessed the short-term and long-term
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safety of borneol on the basis of corneal hydration level, Draize test, electrophysiological, and histological examination. No substantial toxic reactions were observed in corneal hydration level, electrophysiological, or histological examinations after adding the borneol in our colleagues’ studies. Therefore, it can be concluded that the use of borneol is quite safe in the administration of topical drop. In addition, borneol has been widely used for ocular disease in China and the clinical practice has proved the long term clinical safety of low dose borneol at some extent. However, it is necessary to conduct further safety studies before using it clinically. In conclusion, this study evaluated borneol as an ocular drug delivery enhancer for the compounds with different hydrophilicities and molecular sizes. We demonstrated that borneol could enhance transcorneal permeation of both hydrophilic and lipophilic compounds, especially hydrophilic ones. Furthermore, 0.2% borneol can enhance transcorneal permeation of hydrophilic compounds with molecular weight r4 kDa. Additionally, it was nonirritant even at relatively high concentrations. Therefore, borneol can be considered as a safe and effective penetration enhancer for ocular drug administration. Our study indicates the role of borneol, and states that it might be possible to be applied as an adjuvant during the eye treatments. Further studies are needed to confirm the results and to explore the performance of borneol in ophthalmic drug delivery system. References Afouna, M.I., Khedr, A., Abdel-Naim, A.B., Al-Marzoqi, A., 2010. Influence of various concentrations of terpene-4-ol enhancer and carbopol-934 mucoadhesive upon the in vitro ocular transport and the in vivo intraocular pressure lowering effects of dorzolamide ophthalmic formulations using albino rabbits. J. Pharm. Sci. 99, 119–127. Ambati, J., Canakis, C., Miller, J.W., Gragoudas, E., Edwards, A., Weissgold, D.J., Kim, I., Delori, F., Adamis, A.P., 2000. Diffusion of high molecular weight compounds through sclera. Invest. Ophthalmol. Vis. Sci. 41, 1181–1185. Bucolo, C., Drago, F., Salomone, S., 2012. Ocular drug delivery: a clue from nanotechnology. Front. Pharmacol. 3, 188, http://dx.doi.org/10.3389/fphar.2012.00188. Cai, Z., Hou, S., Li, Y., Zhao, B., Yang, Z., Xu, S., Pu, J., 2008. Effect of borneol on the distribution of gastrodin to the brain in mice via oral administration. J. Drug Target. 16, 178–184. Cui, Y., Li, L., Zhang, L., Li, J., Gu, J., Gong, H., Guo, P., Tong, W., 2011. Enhancement and mechanism of transdermal absorption of terpene-induced propranolol hydrochloride. Arch. Pharm. Res. 34, 1477–1485. Davies, N.M., 2000. Biopharmaceutical considerations in topical ocular drug delivery. Clin. Exp. Pharmacol. Physiol. 27, 558–562. Draize, J.H., Woodard, G., Calvery, H.O., 1944. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 82, 377–390.
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