Effects of Labrasol on the corneal drug delivery of baicalin

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Apr 19, 2009 - ISSN 1071-7544 print/ISSN 1521-0464 online © 2009 Informa UK Ltd ..... AAPS Pharm Sci Tech. 7, E1–6. Higuchi, T. ... Drug Dev Ind Pharm.
Drug Delivery, 2009; 16(7): 399–404

RESEARCH ARTICLE

Effects of Labrasol on the corneal drug delivery of baicalin Zhidong Liu1,2, Xinhua Zhang1,2, Jiawei Li3, Rui Liu1,2, Lexin Shu1,2 and Jun Jin4 Research Center of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China, 2Engineering Research Center of Modern Chinese Medicine Discovery and Preparation Technique, Ministry of Education, Tianjin, PR China, 3Department of Experimental Education and 4Department of International Education, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China 1

Abstract Purpose: To investigate the use of Labrasol in ocular drug delivery system. Methods: The in vivo ocular irritation of Labrasol was tested by pathological section observation using rabbits. The effects of Labrasol on corneal permeability of baicalin was investigated in vitro, using isolated rabbit corneas. The pharmacokinetics was evaluated by microdialysis in the rabbit aqueous humors. Results: The results of the ocular irritation studies showed that Labrasol was non-irritant at the concentrations studied (0.5–3.0%(v/v)), while Labrasol produced slight irritation at a concentration of 5.0%(v/v). For the in vitro study, with Labrasol at concentrations of 1.5%, 2.0%, and 3.0%(v/v), the apparent permeability coefficient (Papp) was 1.69-, 3.14-, and 2.23- fold of baicalin solution, respectively. In the pharmacokinetics studies, the AUC value of baicalin solution with 2.0% and 3.0%(v/v) Labrasol were 4.7- and 5.6-fold of that of the control group (p < 0.01), and the Cmax value of baicalin solution with 2.0% and 3.0%(v/v) Labrasol vs the control group were 3.2- and 5.7-fold (p < 0.01). Conclusion: Labrasol may have potential clinical benefits in improving the ocular drug delivery of baicalin. Keywords: Labrasol; corneal permeability; microdialysis; ocular irritation; baicalin

Introduction It is very difficult to achieve an effective concentration of drug within the target area of the eye, because the eye is protected by a series of complex defense mechanisms. For example, the presence of the blood–aqueous barrier, which prevents drugs from entering the aqueous humor, and the blood–retinal barrier, which prevents drugs from entering the extravascular retinal space and the vitreous body (Stjernschantz & Astin, 1993). These complex defense mechanisms lead to a poor bioavailability of drugs delivered in classical ophthalmic dosage forms (eye drops) into the lower cul-de-sac. Drugs systemically administered for their ocular action also have poor access to eye tissue. Drug penetration usually increases with drug lipophilicity until a maximum is reached and then

it ceases to increase and/or decreases with a further increase in drug lipophilicity. A parabolic relationship between the drug penetration rate and its lipophilicity has been observed for the cornea and other biological membranes (Mosher & Mikkelson, 1979; Schoenwald & Huang, 1983; Gershon et al., 1984). Hence, although multiple pathways for drug transport across biological membranes have been proposed, the design of drugs with a suitable lipophilicity is of great importance to optimize drug penetration via the non-polar pathways. Different methods have been used to overcome the barrier property of the cornea; one effective approach is to increase the transcorneal passage of drugs by incorporating penetration enhancers into the formulations. The effects of hexamethylene louramide, hexamethylene octanamide, Azone, and decylmethylsulfoxide on the corneal permeability in vitro were studied (Diane

Address for Correspondence: Zhidong Liu, Research Center of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China. Tel: 86-22-23051965. Fax: 86-22-23050129. E-mail: [email protected] (Received 19 April 2009; revised 06 June 2009; accepted 18 June 2009) ISSN 1071-7544 print/ISSN 1521-0464 online © 2009 Informa UK Ltd DOI: 10.1080/10717540903126165

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et al., 1994). However, the ocular irritation produced by these four penetration enhancers and the ocular pharmacokinetics studies were not studied. Besides, the effects of benzalkonium chloride, EDTA, non-ionic surfactants, surface-active heteroglycoside, and bile salts on the corneal permeability of four -blockers in vitro were studied (Fabrizio et al., 1996). Among these enhancers, sodium deoxycholate, digitonin, escin, and polyoxyethylene 9 lauryl ether had a significant irritant effect at concentrations of 1.0% (w/w), 0.25% (w/w), 0.25% (w/w), and 2.0%(w/w), respectively. Hence, it is very important to carry out ocular irritation studies when employing new penetration enhancers. Besides, the in vitro studies are sometimes different from the in vivo studies, so it is also very important to do the ocular pharmacokinetics studies to verify the result. Labrasol (Caprylocaproyl macrogol-8 glycerides), an oil water-soluble liquid, is extensively used in topical, transdermal, and oral pharmaceutical preparations as an emulsifier and absorption enhancer (Hua et al., 2004; Mori et al., 2004; Fini et al., 2008). When Labrasol was used as the emulsifier to make microemulsion of Acyclovir (Ghosh et al., 2006) after oral administration in rats, the microemulsion showed an absolute bioavailability of 27.83%, which is 12.78-times higher than that of commercially available tablets. Labrasol significantly enhanced the intestinal absorption of rhodamine123 in rats via a passive transport in addition to the inhibitory action for the function of P-gp in the intestine (Lin et al., 2007). To our knowledge, no report has described the use of Labrasol as a penetration enhancer for an ophthalmic drug delivery system. The aim of this study was to explore the use of Labrasol in an ocular drug delivery system. Baicalin is a flavonoid purified from the medicinal plant Scutellaria baicalensis Georgi which has been used for thousands of years in traditional Chinese medicine. It has many significant biological activities to eyes, such as antiinflammatory, antibacterial, and anti-cataract effects (Qi et al., 1998; Cheng et al., 2001). Besides, we chose baicalin as a model compound to study the effect of Labrasol on the corneal permeability and the ocular pharmacokinetics in rabbit eyes. The mechanism of ocular permeation enhancement of drugs by Labrasol was also investigated. In addition, the ocular irritation produced by different concentrations of Labrasol was evaluated.

Materials and methods

Gattefosse (France). All the other chemicals were of analytical grade. Lidocaine hydrochloride injection was purchased from Shanghai Hefeng Pharmaceutical Co. Ltd. (Shanghai, China). Ofloxacin ophthalmic solution was purchased from HuBei Qianjiang Pharmaceutical manufacture (Qianjiang, China). The linear microdialysis probes (MD-2000, 10 mm membrane) were acquired from Bioanalytical Systems Inc. (USA). A microinjection pump was purchased from CMA (Sweden). Animals New Zealand White rabbits, 2.5–3.0 kg, were provided by the Chinese Academy of Medical Sciences of Radiation Research Institute. The animals, housed in standard cages in a light-controlled room at 19 ± 1°C and 50 ± 5% RH, were given a standard pellet diet and water ad libitum. All studies were conducted in accordance with the Principles of Laboratory Animal Care (NIH publication no. 92–93, revised in 1985) and were approved by the Department of Laboratory Animal Research at Tianjin University of Traditional Chinese Medicine. The procedures involving animals were reviewed and approved by the Animal Ethical Committee at Tianjin University of Traditional Chinese Medicine. Ocular irritation studies of Labrasol Ocular irritation studies were performed according to the Draize technique (Draize et al., 1944), which has been used by the FDA to evaluate the safety of several substances (Fitzhugh et al., 1946). A total of 36 rabbits, divided into six groups based on the concentrations of Labrasol, were used. The solutions, consisting of 0.5, 1.0, 1.5, 2.0, 3.0, and 5.0% (v/v) Labrasol in phosphate buffer at pH 7.4, were instilled into the left eyes, and phosphate buffer at pH 7.4 without labrasol into the right eyes, 0.1 mL every 4 h, four times a day for a period of 7 days. The condition of the ocular tissue was monitored 4, 12, 24, 48, and 72 h after the last instillation. The conjunctival congestion, swelling, and discharge were graded on a scale from 0–3, 0–4, and 0–3, respectively. Iris hyperaemia and corneal opacity were graded on a scale from 0–4. The mean values from six treated eyes were calculated for each solution. The evaluation criteria in accordance with the Draize technique were non-irritant from 0–3.9, slightly irritant from 4–8.9, moderately irritant from 9–12.9, and seriously irritant from 13–16. After the in vivo studies, the pathological sections of the eyes were also made.

Materials and animals Materials Baicalin was purchased from ZhongXin Pharmaceutical (> 98%, Tianjin, China). Labrasol was kindly gifted by

Corneal permeation studies After the rabbits were sacrificed by injecting air through marginal ear vein, freshly excised rabbit corneas were

Effects of Labrasol on the corneal drug delivery of baicalin immediately mounted on Franz-type diffusion cells, which were maintained at a constant temperature of 35 ± 1°C, under mixing conditions using a transdermal diffusion machine at a rotating speed of 200 rpm/min (TK-20A, Shaihai, China). The corneal area available for diffusion was 0.50 mm2. Pre-heated (35°C), 0.5 mL of baicalin in phosphate buffer at pH 6.5, with and without labrasol at various concentrations, was added to the epithelial and the phosphate buffer at pH 6.5 was added to the endothelial (4.5 mL) compartment. Samples of medium from the endothelial side were withdrawn every 40 min from the sampling port and were replaced by equal quantity of fresh phosphate buffer at pH 6.5 to maintain a constant volume. Each experiment continued for 4 h and was repeated three times. The apparent corneal permeability coefficients (Papp) were calculated according to equation (1) (Schoenwald & Huang, 1983). Papp = ∆Q/( ∆t × C0 × A × 60) (cm/s)

(1)

where Q/t is the steady-state slope of the linear portion of the plot of the amount of drug in the receiving chamber (Q) vs time (t), A is the area of exposed corneal surface (0.5 mm2), C0 is the initial concentration of drug in the donor cell, and 60 represents the conversion of minutes to seconds. Determination of corneal hydration levels (HL) In order to determine the wet corneal weight (Wa) and the corresponding dry corneal weight (Wb), which was desiccated at 60°C for 16 h, the percentage of corneal hydration level (HL%), defined as [1 – (Wb/Wa)]·100, was determined both for untreated corneas (removed no later than 30 min after the death of the rabbit) and for corneas recovered from permeation tests performed in the absence and presence of enhancers. Measurement of partition coefficients Partition coefficients between n-octanol and aqueous phase were determined by the modified shake-flask method. Briefly, a certain amount of baicalin was dissolved in water with labrasol (0–3.0%(v/v)) and agitated with n-octanol for 24 h. The partition coefficient is the ratio of the drug concentration in the octanol and aqueous phase. Both aqueous phase and n-octanol were saturated with each other before the experiment.

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animals were anesthetized with lidocaine hydrochloride injection. A custom-designed LM-10 microdialysis probe was implanted into the anterior chamber of each eye as described (Lonnroth et al., 1987). Probe inlet and outlet lines were tunneled beneath the conjunctiva, under the upper eyelid, and exited between the ears. The leads were protected with a latex glove pocket affixed to the top of the head. The probe was introduced as described previously (Duchene & Wouessidjewe, 1996). The anchor was sutured to the sclera with 7-0 Vicryl, and conjunctiva was sutured over the anchor. Exterior wound surfaces were treated with ofloxacin 0.3% ophthalmic solution. Animals were used for experimentation after 2 days recovery. Slit-lamp was taken after recovery to estimate fibrin formation and the condition of the eye prior to the use of rabbits in experiments. Conscious rabbits (n = 3) were placed in rabbit restrainers which permitted free movement of the head. Following a 1-h equilibration period with perfusion of saline solution through the probe, different concentrations of standard baicalin saline solutions (0.0516, 0.1032, 0.1548, 0.2064, 0.2580, and 0.3096 g/mL) were perfused through the probe at a rate of 3 L/min, and dialysate were collected for 10 min after 30 min of perfusion. A 20-L aliquot of each fraction was analyzed by HPLC. In vivo recovery was defined as (Higuchi, 1960): R = (Cin – Cout)/(Cm – Cout), where Cin is the concentration of standard solutions; Cout is the concentration of dialysate; and Cm, the concentration in aqueous humor. A linear equation was plotted by (Cin − Cout) vs Cout, and the slope of the line gives the recovery (R). After the disturbance of standard solutions was reduced to the negligible level by the perfusion of saline solution through the probe, 100-L of Baicalin in phosphate buffer at pH 7.4 and that with 2%(v/v) or 3%(v/v) labrasol were dropped in the rabbit eyes. The samples were collected every 10 min in the first hour and 20 min for the remaining hours until baicalin cannot be detected. At the end of the experiment, euthanasia was performed under deep anesthesia with an intravenous injection of sodium pentobarbital through the marginal ear vein (De Lange et al., 2000). Chromatographic analysis Baicalin was assayed by reversed-phase HPLC (Cometro 6000; uSA). The mobile phase is methanol-0.05% formic acid (60:40). The peak area correlating linearly with the concentrations was in the range of 0.0054∼ 0.3240 g/mL (r2 = 0.9999).

Pharmacokinetics studies

Statistical analysis

Rabbits (n = 3) had been treated with ofloxacin 0.3% ophthalmic solution for 4-days before surgery. Then the

The data obtained are expressed as mean ± SD. The ocular irritation data were statistically evaluated by

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Kruskal-Wallis test, and the other experimental data were subjected to statistical analysis t-test. Differences were considered to be significant at p < 0.05.

Results and discussion Ocular irritation studies With regard to the cornea, disperse opacity; iris, hyperaemia; conjunctiva, redness of palpebral conjunctivae, swelling, and discharge with moistening of the lids, Labrasol was non-irritant at concentrations of 0.5–3.0% (v/v), and slightly irritant at 5.0% (v/v). The average scope of ocular irritation test was 0, 0, 0, 1 ± 0.3, 2 ± 0.6, 3 ± 0.7, and 4 ± 0.5 from 0–5% (v/v) Labrasol, respectively. And there were no damage from the pathological section. Based on these results, the concentrations of Labrasol selected for the corneal permeation studies were 0.5, 1.0, 1.5, 2.0, and 3.0% (v/v). Corneal permeation studies Based on the log P (o/w) values listed in Table 1, baicalin was neither lipophilic nor hydrophilic and Labrasol can increase the lipophilicity of baicalin. Figure 1 shows the effect of Labrasol on the Papp values of baicalin. The permeability of baicalin significantly increased as the concentration of Labrasol mounted to 2% and 3%(v/v), the enhancement in Papp was 3.1- and 2.2fold for baicalin (p < 0.01). Concentration of 1.5% (v/v) Labrasol increased the Papp by ∼ 1.7-fold for baicalin (p < 0.05).

The cornea is heterogeneous in structure and can be divided into three layers with different physicochemical properties. The outer epithelium, the most lipophilic, is the major penetration barrier for hydrophilic drugs; the stroma, the most hydrophilic, is the major penetration barrier for lipophilic drugs; and the inner endothelium consists of a single layer of flattened epithelium-like cells. Since the cornea has both hydrophilic and lipophilic structures, it presents an effective barrier to the absorption of both hydrophilic and lipophilic compounds. The results of this study show that Labrasol can increase the corneal penetration of baicalin by producing effect on the corneal barrier functions. Some studies (Kaur & Smitha, 2002) have found that surfactants can incorporate into the lipid bilayer, forming polar defects which can change the physical properties of the cell membrane. When the lipid bilayer is saturated, mixed micelles begin to form, resulting in the removal of phospholipids from the cell membranes and, hence, leading to membrane solubilization. Labrasol is a surfactant and the results suggest that the mechanism of Labrasol on baicalin corneal transport may involve changes in the structure of the epithelium because Labrasol produces micelles in the epithelial lipid bilayer, which can result in the removal of phospholipids from the epithelial cell membranes and finally lead to the increase in the transcorneal passage of baicalin. Effect of corneal hydration levels The percentage corneal hydration is a parameter frequently used to evaluate damage to this tissue. As

40 30 20 10 0

1.5 −0.3068 22.3* ± 2.88 23.6* ± 3.04

0.00 0.0 −0.02

50 Cout − Cin/(µg·mL−1)

Corneal permeability coefficient × 107/(cm·s−1)

Table 1. Effect of Labrasol on the corneal permeability of baicalin in vitro (n = 3). Labrasol/%(v/v) 0 0.5 1.0 Log P −0.7565 −0.6379 −0.5010 13.2 ± 2.40 14.8 ± 1.57 19.5 ± 3.70 Papp × 107/cm/s 13.8 ± 2.51 15.6 ± 1.66 20.6* ± 3.91 Jss × 105/(g/s cm2) * p < 0.05; ** p < 0.01 vs control.

0.1

2.0 −0.1150 41.5** ± 7.05 40.7** ± 6.91

0.2

0.3

3.0 −0.2562 29.5** ± 2.35 29.2* ± 2.32

0.4

−0.04 −0.06 −0.08

Cout − Cin = −0.4441 × Cin − 0.0011 R2 = 0.9962

−0.10 −0.12 −0.14

0

0.5

1

1.5

2

3

−0.16

Cin/(µg·mL−1)

Labrasol% (V/V)

Figure 1. The corneal permeability coefficients of Baicalin (n = 3).

Figure 2. In vivo recovery of microdialysis probe in aqueous humor (n = 3).

Effects of Labrasol on the corneal drug delivery of baicalin Table 2. The corneal hydration of baicalin in the presence and absence of Labrasol (n = 3). Labrasol/%(v/v) 0 0.5 1.0 1.5 Corneal hydration% (M ± SD) 80.0 ± 1.86 81.7 ± 0.63 80.95 ± 1.31 81.17 ± 1.40

2.0 79.45 ± 1.21

Table 3. Pharmacokinetics parameters of baicalin in aqueous humor after topical administration in conscious rabbit (n = 3). Tmax/h Ke/h−1 Drug AUC/(g/mL·h) Cmax (g/mL) Reference 0.0269 ± 0.0031 2%Labrasol 0.1265 ± 0.0361** 3%Labrasol 0.1509 ± 0.0703** * p < 0.05; ** p < 0.01 vs control.

0.0621 ± 0.0060 0.2008 ± 0.0219** 0.3539 ± 0.0038**

0.5

C/(µg·mL−1)

0.4 0.3 0.2 0.1 0 0.0

0.3

0.7

1.0

1.7

2.3

t/h

Figure 3. Aqueous humor Baicalin concentration-time profiles following a 100-L topical dose in conscious rabbits (n = 3).

reported (Maurice & Riley, 1970), the normal cornea has a hydration level of 76–80%. An 83–92% hydration level denotes damages to the epithelium or endothelium. As shown in Table 2, the percentage corneal hydration of drugs after the studies was not higher than 83%. This indicates that the Labrasol did not cause any damage to the epithelium or endothelium during the studies. Pharmacokinetics studies As is shown in Figure 2, the linear regression between perfusate (Cin) and dialysate (Cout): Cout − Cin = −0.4441Cin − 0.0011 (R2 = 0.9962), so the recovery (r) in vivo is 44.41 ± 6.49%. In vitro, perfusion flow-rate, temperature, perfusate composition, characteristics of the drug, characteristics of the semi-permeable membrane, and the surface of the semi-permeable membrane may all affect recovery. All parameters that influence recovery in vitro will also influence recovery in vivo. However, in vivo, tissue characteristics will play an important role and may ultimately determine the recovery. Recovery in vivo depends on the diffusion in three regions: probe lumen, dialysis membrane, and periprobe environment (Bungay et al., 1990; Benveniste et al., 1991). Diffusion in probe lumen is limiting only

0.278 ± 0.096 0.167 ± 0 0.167 ± 0

2.8597 ± 1.6652 1.7880 ± 0.8503 1.3717 ± 0.8642

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3.0 78.98 ± 0.59

t1/2/h 0.3639 ± 0.3147 0.4408 ± 0.1690 0.6467 ± 0.3537

with the use of very low flow rates. Diffusion through the dialysis membrane is limiting only when transport through the periprobe environment is rapid. Rapid diffusion through the periprobe environment occurs in most flowing systems. In tissues, effective diffusion through the extracellular fluid determines the recovery of the microdialysis probes (Morrison et al., 1991). Aqueous humor pharmacokinetic parameters were presented in Table 3. As is shown in Figure 3, the AUC of baicalin solution with 2.0% and 3.0%(v/v) Labrasol were much bigger than the the reference group, they were 4.7- and 5.6-fold vs the reference group (p < 0.01), and the Cmax values of baicalin solution with 2.0% and 3.0%(v/v) Labrasol vs the control group were 3.2- and 5.7-fold (p < 0.01). The Tmax values of test group is shorter than that of control group, and Ke values of test group is higher than that of control group.

Conclusion This study evaluted the suitability and feasibility of using Labrasol as an ocular drug delivery system. The results of the ocular irritation studies indicate that Labrasol is non-irritant at a concentration ranging from 0.5–3.0% (v/v). Besides, none of these concentrations caused visible ocular damage or abnormal clinical signs involving the cornea, iris, and conjunctiva. In vitro and in vivo studies prove that Labrasol can increase the permeability and bioavailability of baicalin in ocular drug delivery. Therefore, Labrasol may have potential clinical benefits in improving the ocular drug delivery.

Acknowledgement This study was supported by International Science Cooperation Project (No2007DFC 31670, China). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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