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Effect of ion-pairing on the permeation of glibenclamide through rat skin
J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang*, X.C. Niu, Y.X. Jiang, Z.G. He Department of Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning, 110016, China *Correspondence: [email protected] The purpose of the present study was to investigate the effect of ion-pairing on the permeation of glibenclamide through rat skin. Diethylamine, triethylamine, ethanolamine, diethanolamine and triethanolamine were used as counter-ions. The steady-state flux of glibenclamide markedly increased in the presence of counter-ions (P < 0.05). These results suggest that it is possible to enhance the permeation of glibenclamide by using an ion-pair approach. Moreover, the extent of enhancement possibly depends on the alkalinity, molecular weight, structure of the counter-ions and the dielectric constant of the donor solution. The influence of penetration enhancers on the permeation of glibenclamide was also determined. All the penetration enhancers studied increased the flux of glibenclamide compared with the control. Among these enhancers, menthol and N-methyl2-pyrrolidone showed the greatest enhancing activity. Key words: Glibenclamide – Ion-pairing – Penetration enhancers – Percutaneous absorption.
Glibenclamide is a potent, second-generation oral sulfonylurea hypoglycemic agent widely used in the treatment of non insulindependent diabetes. It lowers blood glucose levels by stimulating depolarization of pancreatic beta cells, therefore inducing the release of endogenous insulin [1]. However, many sulfonylureas have been associated with severe and sometimes fatal hypoglycemia and gastric disturbances like nausea, vomiting, heartburn, anorexia and increased appetite after oral therapy [2]. Since sulfonylureas are usually taken for a long period, the compliance of the patients is very important [3]. Due to the side effects after oral administration, the development of new preparations of glibenclamide is of significant importance. The transdermal route would be an attractive alternative for systemic delivery of glibenclamide, because it offers several advantages over conventional routes: avoidance of first-pass metabolism, the production of relatively constant drug plasma levels, a concurrent reduction in side effects, relative ease of drug input termination, and improved patient compliance. Nevertheless, the barrier function of the skin in protecting the body from physical and chemical attack makes delivery of the required drug dose through the skin and to the target organ difficult. A number of potential methods to enhance skin transport of drugs have been proposed, e.g. penetration enhancers, ion pair formation, prodrug design, iontophoresis, phonophoresis, and electroporation and some of them, including penetration enhancers and iontophoresis, have already been applied to the skin transport of glibenclamide [4-6]. Ion-pairs are defined as neutral species formed only by electrostatic attraction between oppositely charged ions [7], which are sufficiently lipophilic to dissolve in a lipoidal medium, such as the stratum corneum. The formation of ion-pairs to increase the skin penetration of drugs has been reported [8-10]. For example, Megwa et al. showed that secondary, tertiary and quaternary amines increased the in vitro permeation of salicylate across the human epidermis [11, 12]. Fang et al. found that the skin permeation of mefenamic acid increased in the presence of diethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine and propanolamine [13]. Sarveiya et al. reported increased penetration of ibuprofen through a polydimethylsiloxane membrane following ion-pair formation with alkylamines [14]. However, no information is available on the skin transport of glibenclamide following ion-pair formation. The objective of this study was to investigate the effect of ionpairing on the permeation of glibenclamide. In addition, the use of
penetration enhancers is a long-standing and widely used approach to increase transdermal and topical delivery [15]. In order to compare the permeation-enhancing effect of counter-ions with that of penetration enhancers, the influence of penetration enhancers on the permeation of glibenclamide was also evaluated.
I. Materials and methods 1. Materials
Glibenclamide (GLB) was purchased from Shandong Boshan Pharmaceuticals Co., Ltd. (Shandong, China). Diethylamine (DEA), triethylamine (TEA), ethanolamine (EtA), diethanolamine (DEtA), triethanolamine (TEtA), oleic acid and polyethylene glycol 400 (PEG400) were all supplied by Tianjin Bodi Chemicals Co., Ltd. (Tianjin, China). Methanol was of HPLC grade and purchased from Shandong Yuwang Chemicals Co., Ltd. (Shandong, China). Ethanol (EtOH), isopropyl myristate (IPM), N-methyl-2-pyrrolidone (NMP), azone and menthol were obtained from Tianjin Baishi Chemicals Co., Ltd. (Tianjin, China), China National Medicines Co., Ltd. (Shanghai, China), Beijing Chemical Industry (Beijing, China), Tianmen Kejie Pharmaceuticals Co., Ltd. (Hubei, China) and Chengdu Kelong Chemical Industry (Sichuan, China), respectively. All other chemicals and solvents were of analytical grade.
2. Preparation of skin samples
Male Wistar rats weighing 180-220 g (6-8 weeks old) used in all experiments were supplied by the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). The experiments were performed in accordance with the guidelines for animal use in the Life Science Research Center of Shenyang Pharmaceutical University. The rats were anesthetized with 20% urethane and then the hair from their abdomen was removed using animal hair clippers (model 900, TGC, Japan). Full thickness abdominal skin was harvested immediately after sacrificing the animals. The subcutaneous tissue and fat were carefully removed with surgical scissors, then the skin was washed with normal saline, wrapped in plastic pockets and stored at -20°C until use (within 1 week). The skin membrane was checked to ensure that no obvious defects were present.
3. In vitro permeation studies
The in vitro skin permeation experiments were carried out using
279
J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
a side-by-side (2-chamber) diffusion cell having a volume of 2.5 mL and an effective diffusion area of 0.95 cm2. A piece of full-thickness rat skin was mounted between the two half cells and fastened with a spring clamp. The dermis side of the skin was in contact with the receiver compartment and the stratum corneum with the donor compartment. IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w) were used as donor solutions. The receiver compartment was filled with 2.5 mL pH 7.4 phosphate buffer containing 40% PEG 400 and the donor compartment with the drug suspension (with and without 5% enhancers or counter-ions at the same molar concentration as GLB). During all the experiments, excess drug was maintained in the donor compartment. Both compartments were then stirred at 600 rpm with a star-head bar driven by a synchronous motor and maintained at 32°C via water flow through a water jacket surrounding the cell. Samples (each 2 mL) were withdrawn from the receiver solution every 2 h over a period of 10 h and replaced with an equal volume of fresh receiver solution to maintain sink conditions. The experiments were performed at least in triplicate. The concentration of drug in the samples was analyzed, and the cumulative amount was plotted against time. The pseudo steady-state flux and lag time were determined as the slope of the linear portion of the plot and the intercept obtained by extrapolation of the linear portion to the time axis, respectively, and the permeability coefficient (P) was calculated by dividing the flux by the drug concentration in the donor phase. The enhancement ratio (ER) was calculated by dividing the cumulative amount of GLB permeated after 10 h (Q10) obtained with the strategies applied (penetration enhancers, counter-ions) by that of the control [16]. Statistical analysis was performed with Student’s t-test and a p-value of 0.05 or less was considered significant.
ben was used as the internal standard. The detector wavelength was 230 nm and the injection volume was 20 µL. The retention times of propylparaben and GLB were 5.2 and 8.5 min, respectively.
II. Results and discussion 1. Effect of ion-pairing on the permeation of GLB
In order to determine whether the polarity of the vehicle could influence the permeation of GLB in the presence of counter-ions through rat skin, permeation experiments were performed using IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w) as donor solutions. The effect of counter-ions on the permeation of GLB through rat skin from vehicle 1 (IPM: EtOH, 3:7) and 2 (IPM: EtOH, 7:3) is shown in Figure 1. The corresponding permeation parameters are presented in Table II. As seen in Table II, GLB was poorly soluble in both vehicles, and the addition of all counter-ions, except TetA, increased the solubility significantly (P < 0.05). For both vehicles, the steady-state flux of GLB in the presence of counter-ions was significantly higher (P < 0.05) than the control. The GLB flux observed with vehicle 1 follows the order: DEA ≈ TEA > DEtA > EtA > TEtA > control. DEA increased the Q10 by 7.24-fold, while TEA, DEtA, EtA and TEtA increased it by a factor of 7.19, 5.39, 2.90 and 2.41, respectively. There was no statistically significant difference between the enhancement produced by DEA and TEA. The flux observed with vehicle 2 was in the order of: EtA > DEA > TEA > DEtA > TEtA > control. EtA increased the Q10 by 41.58-fold, while DEA, TEA, DEtA and TEtA increased it by a factor of 28.87, 23.01, 12.15 and 5.44, respectively. No significant difference (P < 0.05) was observed between the Q10 of DEA and TEA. The formation of an ion-pair between GLB and counter-ions may be responsible for the enhanced skin permeation of GLB. In addition, the enhancing effect of all counter ions, except TEtA, can be attributed in part to the increased solubility of GLB in the donor phase. This may be explained by the simple equation for steady-state flux (Equation 1). When we plot the cumulative mass of diffusant, m, passing per unit area through a membrane, at long times the graph approaches linearity and its slope yields the steady flux, dm/ dt, as in the following equation [17]:
4. Analytical method
The concentration of GLB was determined by HPLC. The HPLC system consisted of a pump (HITACHI L-7100), a UV-Vis Detector (L-7420), a column temperature controller (HT-220A, Tianjin, China), a data station (T-2000L Tianmei Techcom, China), and a 5-µm ODS column (200 mm × 4.6 mm, DIKMA Technologies). The mobile phase was methanol - water (73:27, v/v) adjusted to pH 3.5 with phosphoric acid, and this was delivered at a flow rate of 1.0 mL/min. Propylpara-
(dm/dt) = (DC0K/b)
where C0 represents the constant donor drug concentration; K, the partition coefficient of solute between membrane and bathing solution; D, the diffusion coefficient; and b, the membrane thickness. From Equation 1, we can conclude that the flux may be increased by increasing the solubility of drug in the donor solution. Bjerrum’s equation [9], which describes a critical separation distance for the formation of an ion-pair, highlights the importance of the dielectric constant (e): a solvent with a high dielectric constant such as water (e = 78.5) is unfavorable for ion-pair formation, while the interaction becomes increasingly important in solvents with e < 40
Table I - Composition of the donor solutions. Vehicle
IPM (g)
EtOH (g)
GLB (mol)
Counter ions (mol)
Enhancers (g)
1
30 30 30
70 70 70
0.0081 0.0081 0.0081
0.0081 -
5
2
70 70 70
30 30 30
0.0081 0.0081 0.0081
0.0081 -
5
Eq. 1
Table II - Permeation parameters of GLB in the presence of various counter ions through rat skin from IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w) (mean ± SE of three experiments). Vehicle
Counter ion
Solubility (mg/mL)
Flux (µg/cm2/h)
Lag time (h)
ER
P x 103 (cm/h)
IPM:EtOH (3:7, w/w)
control DEA TEA EtA DEtA TEtA
1.17 2.84 4.46 12.00 6.98 0.32
9.64 ± 0.42 61.00 ± 9.13 59.35 ± 9.13 29.42 ± 2.81 51.80 ± 4.62 25.67 ± 4.34
3.73 ± 0.16 2.64 ± 0.98 2.65 ± 0.68 4.00 ± 0.20 3.74 ± 0.37 4.23 ± 0.48
7.24 ± 0.92 7.19 ± 1.34 2.90 ± 0.20 5.39 ± 0.72 2.41 ± 0.32
8.24 ± 0.36 21.49 ± 3.22 13.32 ± 2.05 2.45 ± 0.23 7.42 ± 0.66 79.95 ± 13.53
IPM:EtOH (7:3, w/w)
control DEA TEA EtA DEtA TEtA
0.77 1.11 2.56 3.50 5.36 0.68
10.09 ± 1.03 280.08 ± 39.71 272.72 ± 54.94 507.64 ± 28.46 136.75 ± 9.97 57.78 ± 4.57
3.92 ± 0.23 3.53 ± 0.38 4.95 ± 0.44 5.03 ± 0.18 4.67 ± 0.26 4.14 ± 0.50
28.87 ± 3.15 23.01 ± 5.38 41.58 ± 2.72 12.15 ± 1.03 5.44 ± 0.22
13.07 ± 1.34 252.55 ± 35.80 106.62 ± 21.48 145.00 ± 8.13 25.49 ± 1.86 84.74 ± 6.70
280
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
favorable for ion-pair formation. This suggests that the ion-pair skin transport of GLB follows Bjerrum’s equation. An increase in the lag time was observed when the concentration of IPM increased from 30 to 70%, indicating a certain reduction in the diffusion ability of GLB. This also suggests that EtOH is more potent in increasing the diffusion ability of GLB than IPM. Our results are in accordance with another report, where the diffusion ability (diffusion parameter) of emedastine decreased as the concentration of IPM increased from 27 to 63% [18]. In addition, the permeability coefficient of GLB increased markedly as the concentration of IPM increased from 30 to 70%. The permeability coefficient is equal to the product of the partition coefficient and the diffusivity of the drug in the membrane divided by the thickness of the membrane (assumed to be relatively constant) [23]. The increase in the permeability coefficient may therefore be due to the increase in the partition coefficient. The physicochemical properties of various counter-ions are summarized in Table III. It was observed that some properties of counterions could affect their permeation-enhancing effect significantly. The cumulative amount of GLB was found to increase as a function of the pKa value of the counter-ion except for EtA (Figure 2). A high pKa
(A) IPM:EtOH (3:7, w/w)
600
Cumulative amount permeated (?g/cm≤)
500
400
300
200
100
0 0
2
4
6
8
10
Time (h)
(B) IPM:EtOH (7:3, w/w)
3000
Table III - Physicochemical properties of various counter ions (Data were obtained from the SRC PhysProp Database).
2500
2000
1500
1000
Counter ion
pKa
log KO/W
Molecular weight
DEA TEA EtA DEtA TEtA
11.1 10.8 9.5 8.96 7.76
0.58 1.45 -1.31 -1.43 -1.00
73.14 101.19 61.08 105.14 149.19
(A) IPM:EtOH (3:7, w/w)
500 600
0
y = 103.76x - 566.02 2
R = 0.9196
500
0
2
4
6
8
10 Q (µg/cm2)
Cumulative amount permeated (?g/cm≤)
J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
Time (h)
Figure 1 - The effect of various counter-ions on the permeation of GLB through rat skin from IPM:EtOH (3:7, w/w) (A) and IPM:EtOH (7:3, w/w) (B). Each point represents the mean ± S.E. of three experiments. Key: (◊) control; (u) TEtA; (D) EtA; (s) DEtA; (p) DEA; (n) TEA.
400 300 200 100
[7]. A lipophilic system consisting of IPM and EtOH (3:7 or 7:3) was employed in this study. The dielectric constant of IPM and EtOH is 3.31 and 24.13, respectively [18]. To determine the significance of ion-pairing on the permeation of ionic drugs, phosphate buffer [8, 9], McIlvaine’s buffer [19], water-propylene glycol (70:30, w/w) [20], EtOH-pH 6.4 buffer solution (1:2, v/v) [10], propylene glycol (PG) [14, 21, 22] and EtOH-PG (2:1, v/v) [11, 12] have been used as donor solutions. PG is a solvent with a relatively low dielectric constant (e = 32.1) [22]. Compared with these donor solutions, the dielectric constant of the IPM/EtOH system is the lowest. Therefore, the IPM/ EtOH system is suitable for evaluating the effect of ion-pairing on the percutaneous absorption of ionic drugs. As shown in Table II, the flux of GLB in the presence of counter-ions increased significantly (P < 0.05) as the concentration of IPM increased from 30% to 70%. The dielectric constants of IPM:EtOH (27:73, v/v) and IPM:EtOH (63:37, v/v) are 18.36 and 9.13, respectively [18]. Since the relative percentage of IPM and EtOH in vehicle 1 and IPM:EtOH (27:73, v/v) is similar, the dielectric constant of vehicle 1 is similar to that of IPM:EtOH (27:73, v/v). Likewise, the dielectric constant of vehicle 2 is comparable to that of IPM:EtOH (63:37, v/v). The dielectric constant of vehicle 2 was lower than that of vehicle 1. The flux of GLB in the presence of counter-ions increased as the dielectric constant of the vehicle decreased, indicating that a vehicle with a low dielectric constant is
7
8
9
10
11
12
p Ka
(B) IPM:EtOH (7:3, w/w) 2000
y = 409.13x - 2873.1 2
Q (µg/cm2)
R = 0.9752 1500
1000
500
0 7
8
9
10
11
12
p Ka
Figure 2 - Relationship between the cumulative amount (Q) of GLB in the presence of counter-ions, except EtA, from IPM:EtOH (3:7, w/w) (A) and IPM:EtOH (7:3, w/w) (B) and the pKa of the counter-ions. Each point represents the mean of three experiments. 281
J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
generally results in a high cumulative amount. The present results indicate that the enhancing effect of counter-ions is related to their alkalinity. The model of Huyskens and Zeegers-Huyskens predicts that a difference of 2.46-5.8 orders of magnitude between the acid dissociation constants of the base (pKa for DEA, TEA, EtA, DEtA, TEtA are 11.1, 10.8, 9.5, 8.96 and 7.76, respectively) and the acid (GLB, pKa = 5.3) leads to an almost complete shift of the proton-transfer equilibrium of the O-H···N ↔ O-···H-N+ system [24]. The larger the difference between the pKa of the base and acid, the larger the attractive force between the base and acid. In general, some structure activity relationships were apparent from our results in that the counter-ions with a hydroxyl functional group were less potent enhancers than those without a hydroxyl. Moreover, for vehicle 2, the flux of GLB decreased as the number of hydroxyls of the alkanolamine increased, indicating that the hydroxyl group has a negative effect on the permeation of GLB. It has been reported that oxygen-containing monoterpenes may preferentially form hydrogen bonds with ceramide head groups of stratum corneum [25, 26]. Likewise, alkanolamine with a hydroxyl group may also form hydrogen bonds with ceramide head groups. Potts and Guy found that hydrogen bonding ability had a negative effect on drug transport across the skin [27]. Therefore, the hydroxyl group may inhibit the permeation of ion pairs by forming hydrogen bonds with ceramide head groups. In addition, for vehicle 2, the cumulative amount of GLB increased with a decrease in the counter-ion molecular volume, expressed as a molecular weight (for many compounds, the molecular weight is often a reasonable approximation of the molecular volume) (Figure 3). For example, EtA, with the highest cumulative amount, has the lowest molecular weight. In contrast, TEtA, with the lowest cumulative amount, has a relatively high molecular weight. As the molecular weight of the counter-ions decreases, the cumulative amount of GLB increases to give an inverse relationship between the cumulative amount and the molecular weight. The importance of the molecular size of the counter-ions for the penetration of cephalexin through rat skin has been emphasized by Hatanaka et al. [9]. Megwa et al. also observed a close dependence of the flux of salicylate on the molecular size of the amine counter-ions [11]. Ion-pairs with small counter-ions are believed to have a small volume and, thus, may easily pass through the skin. However, for vehicle 1, there is no linear relationship between the cumulative amount of GLB and the molecular weight of counter-ions. This may be due to the fact that vehicle 1 with a higher EtOH concentration has a greater solvent drag effect of EtOH which probably obscures the smaller size effect. The flux of GLB does not correlate with the lipophilicity of the counter-ions expressed as logKO/W. For vehicle 2, the highest flux was measured with EtA. The highest ability to partition into n-octanol was measured with TEA but
2. Effect of penetration enhancers on the permeation of GLB
The effect of penetration enhancers on the permeation of GLB through rat skin from vehicle 1 and 2 is shown in Figure 4. The corresponding permeation parameters are summarized in Table IV. As can be seen from Table IV, the solubility of GLB in both vehicles increased significantly (P < 0.05) when penetration enhancers were used. For vehicle 1, only oleic acid did not produce a significant increase in the flux of GLB (P < 0.05). In all the other cases, the flux
Cumulative amount permeated (µg/cm2)
100
50
0
2
4
6
8
10
Time (h) 250
2
Log Q (µg/cm2)
150
0
y = -0.0099x + 4.011 R = 0.9363
3.5
(A) IPM:EtOH (3:7, w/w)
200
Cumulative amount permeated (µg/cm2)
4
the flux was 1.9-fold lower than that observed with EtA. Although the partition coefficient (logKO/W) of EtA is similar to that of DEtA, the flux of GLB in the presence of EtA is 3.7-fold higher than that in the presence of the latter. Similar results have been reported for lignocaine [8]. Among the counter-ions examined, TEtA exhibited the lowest penetration- enhancing effect in the present study. There may be several explanations for this. The solubility of GLB was the lowest in the presence of TEtA. In addition, the formation of ion pairs is only possible if the ions approach each other and reach a critical separation distance [7]. However, the tertiary alkanol group of TEtA may make it difficult for the nitrogen atom of TEtA to approach the GLB molecule.
3
2.5
(B) IPM:EtOH (7:3, w/w)
200
150
100
50
0 2
0 40
60
80
100
120
140
2
4
6
8
10
160
Time (h) MW
Figure 4 - In vitro permeation profiles of GLB in the presence of various penetration enhancers through rat skin from IPM:EtOH (3:7, w/w) (A) and IPM:EtOH (7:3, w/w) (B). Each point represents the mean ± SE of three experiments. Key: (◊) control; (u) oleic acid; (D) azone; (s) NMP; (p) menthol.
Figure 3 - Relationship between the logarithm of the cumulative amount (Q) of GLB in the presence of counter-ions from IPM:EtOH (7:3, w/w) and the molecular weight (MW) of the counter-ions. Each point represents the mean of three experiments. 282
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
of GLB showed statistically significant differences with respect to the control (P < 0.05). Menthol provided the highest increase in flux, followed by Azone, NMP and oleic acid. Similar to the flux, the highest increase in Q10 was observed with menthol, which increased the Q10 by 2.24-fold, followed by NMP (1.45-fold), azone (1.45-fold), and oleic acid (1.08-fold). Azone and menthol significantly shortened the lag time of GLB compared with the control (P < 0.05). For vehicle 2, all the penetration enhancers tested significantly enhanced the flux of GLB in comparison to the control (P < 0.05). NMP provided the highest increase in flux, followed by menthol, azone and oleic acid. However, the highest increase in Q10 was observed with menthol, which increased the Q10 by 3.06-fold, followed by NMP (2.89-fold), oleic acid (1.86-fold), and azone (1.77-fold). There was a significant decrease in the lag time (P < 0.05) of GLB by menthol when compared with the control. The permeation-enhancing effect of enhancers may be attributed partially to the increased solubility of GLB in the donor phase. As shown in Table III, the increase in EtOH concentration in vehicle from 30 to 70% (w/w) results in a slight decrease in flux. Megrab et al. reported that the decreased permeation of a lipophilic drug (estradiol) from vehicles with higher EtOH concentrations was due to dehydration of the stratum corneum [28]. The decrease in the permeability of GLB through rat skin may also be due to dehydration of the lamellar layers of the stratum corneum. The solubility of GLB in vehicle 1 was significantly higher (P < 0.05) than that in vehicle 2, indicating that EtOH has a greater ability to dissolve GLB compared with IPM. Considering the results obtained with vehicle 2, it seems that there is an inverse relationship between the lipophilicity of enhancers expressed as logKO/W (n-octanol/water partition coefficient) and their enhancing activity (Figure 5). The highest enhancing effect was obtained with NMP and menthol, compounds with logKO/W values of -0.38 and 3.40, whereas more lipophilic compounds, such as Azone (logKO/W = 6.28) and oleic acid (logKO/W = 7.73), had lower capacity to increase the flux of GLB. However, Femenía-Font et al. found that there is an optimum lipophilicity of the promoting agent as far as enhancing activity is concerned [29]. This discrepancy was due to the different vehicles, barriers and drugs employed in the two studies. As logKO/W increases, the enhancer becomes more like the vehicle (the enhancer should be more soluble in the vehicle). For enhancers with a low logKO/W, the interaction between enhancers and the stratum corneum will probably be strong because the vehicle has a weak affinity for enhancers. Conversely, for enhancers with a high logKO/W, the vehicle has a strong affinity for enhancers and, therefore, the interaction will be weak. Thus, enhancers with a high lipophilicity exhibit low enhancing activity. In contrast, the enhancing effect of enhancers with a low lipophilicity should be high. However, for vehicle 1, we could not obtain a good linear correlation of the lipophilicity of enhancers with their enhancing activity. The lipophilicity of vehicle 1 is lower than that of vehicle 2. For enhancers with a low logKO/W, the interaction between enhancers and the stratum corneum will probably be weaker because vehicle 1 has a stronger affinity for enhancers compared to
J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
y = -1.5989x + 29.287
35
2
R = 0.9413
30
Flux (µg/cm2/h)
25 20 15 10 5 0 -1
0
1
2
3
4
5
6
7
8
Log K o/w
Figure 5 - Relationship between the fluxes of GLB in the presence of various penetration enhancers from IPM:EtOH (7:3, w/w) and the noctanol/water partition coefficient (logKO/W) of the penetration enhancers. Each point represents the mean of three experiments.
vehicle 2. Conversely, for enhancers with a high logKO/W, the vehicle has a weaker affinity for enhancers and, therefore, the interaction will be stronger compared to vehicle 2. Thus, the lipophilicity dependency of vehicle 1 is much less than that of vehicle 2. The enhancing effect of the counter ions is greater than that of penetration enhancers. Some researchers reported that the viable epidermis, not the stratum corneum, is the rate-limiting barrier for the transport of very lipophilic chemicals [18, 23, 30]. Since GLB is a lipophilic drug, the viable epidermis is the rate-limiting step in skin penetration rather than the stratum corneum. Penetration enhancers increase the transdermal drug penetration by altering the barrier function of the stratum corneum. Counter-ions may decrease the lipophilicity of GLB by forming an ion-pair and hence may improve the partition of GLB into the viable epidermis. The penetration of GLB is determined by the viable epidermis and the counter-ions are therefore more effective than penetration enhancers for increasing the permeation of GLB through rat skin. * The results of this study suggest that it is possible to increase the permeation of GLB by using an ion-pair approach. Counter-ions are more efficient than penetration enhancers for increasing the permeation of GLB through rat skin. Moreover, the degree of enhancement possibly depends on the alkalinity, molecular weight, structure of the counter-ions and the dielectric constant of the donor solution. Among the penetration enhancers examined, menthol and NMP showed the greatest enhancement activity, indicating that they could be promising penetration enhancers for transdermal delivery systems for GLB.
Table IV - Permeation parameters of GLB in the presence of various penetration enhancers through rat skin from IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w). Vehicle
Enhancers
Solubility (mg/mL)
Flux (µg/cm2/h)
Lag time (h)
ER
P x 103 (cm/h)
IPM:EtOH (3:7, w/w)
control NMP azone oleic acid menthol
1.17 2.06 1.62 1.61 1.42
9.64 ± 0.42 12.79 ± 1.63 13.46 ± 0.16 10.15 ± 2.10 18.86 ± 3.04
3.73 ± 0.16 3.13 ± 0.51 3.45 ± 0.11 3.45 ± 0.40 2.73 ± 0.07
1.45 ± 0.21 1.45 ± 0.03 1.08 ± 0.19 2.24 ± 0.35
8.24 ± 0.36 6.21 ± 0.79 8.32 ± 0.10 6.32 ± 1.31 13.32 ± 2.15
IPM:EtOH (7:3, w/w)
control NMP azone oleic acid menthol
0.77 1.69 1.03 0.90 1.09
10.09 ± 1.03 29.03 ± 1.75 18.09 ± 3.23 16.94 ± 3.35 25.86 ± 5.40
3.92 ± 0.23 3.86 ± 0.19 4.00 ± 0.40 3.06 ± 0.53 2.56 ± 0.43
2.89 ± 0.08 1.77 ± 0.31 1.86 ± 0.26 3.06 ± 0.48
13.07 ± 1.34 17.21 ± 1.04 17.56 ± 3.14 18.86 ± 3.73 23.68 ± 4.94
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Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
14. 15. 16.
17. 18.
19.
Gerich J.E. - Oral hypoglycemic agents. - N. Engl. J. Med., 321, 1231-1245, 1989. Ikegami H., Shima K., Tanaka Y., Hirota M. - Interindividual variation in the absorption of glibenclamide in man. - Acta. Endocrinol. (Copenh.), 111, 528, 1986. Takahashi Y., Furuya K., Iwata M., Onishi H., Machida Y., Shirotake S. - Trial for transdermal administration of sulfonylureas. - Yakugaku Zassi, 117, 1022-1027, 1997. Mutalik S., Udupa N. - Transdermal delivery of glibenclamide and glipizide: in vitro permeation studies through mouse skin. - Pharmazie., 57, 838-841, 2002. Mutalik S., Udupa N. - Effect of some penetration enhancers on the permeation of glibenclamide and glipizide through mouse skin. - Pharmazie., 58, 891-894, 2003. Takahashi Y., Iwata M., Machida Y. - Enhancing effect of switching iontophoresis on transdermal absorption of glibenclamide. - Yakugaku Zasshi, 121, 161-166, 2001. Kraus C.A. - The ion pair concept: Its evolution and some applications. - J. Phys. Chem., 60,129-141, 1956. Valenta C., Siman U., Kratzel M., Hadgraft J. - The dermal delivery of lignocaine: influence of ion pairing. - Int. J. Pharm., 197, 77-85, 2000. Hatanaka T., Kamon T., Morigaki S., Katayama K., Koizumi T. - Ion pair skin transport of a zwitterionic drug, cephalexin. - J. Control. Release, 66, 63-71, 2000. Trotta M., Ugazio E., Peira E., Pulitano C. - Influence of ion pairing on topical delivery of retinoic acid from microemulsions. - J. Control. Release, 86, 315-321, 2003. Megwa S.A., Cross S.E., Benson H.A.E., Roberts M.S. - Ion-pair formation as a strategy to enhance topical delivery of salicylic acid. - J. Pharm. Pharmacol., 52, 919-928, 2000. Megwa S.A., Cross S.E., Whitehouse M.W., Benson H.A.E., Roberts M.S. - Effect of ion pairing with alkylamines on the in-vitro dermal penetration and local tissue disposition of salicylates. - J. Pharm. Pharmacol., 52, 929-940, 2000. Fang L., Kobayashi Y., Numajiri S., Kobayashi D., Sugibayashi K., Morimoto Y. - The enhancing effect of a triethanolamineethanol-isopropyl myristate mixed system on the skin permeation of acidic drugs. - Biol. Pharm. Bull., 25, 1339-1344, 2002. Sarveiya V., Templeton J.F., Benson H.A.E. - Ion-pairs of ibuprofen: increased membrane diffusion. - J. Pharm. Pharmacol., 56, 717-724, 2004. Moster K., Kriwet K., Naik A., Kalia Y.N., Guy R.H. - Passive skin penetration enhancement and its quantification in vitro. - Eur. J. Pharm. Biopharm., 52, 103-112, 2001. Kim N., El-Kattan A.F., Asbill C.S., Kennette R.J., Sowell J.W.Sr., Latour R., Michniak B.B. - Evaluation of derivatives of 3-(2-oxo-1-pyrrolidine) hexahydro-1H-azepine-2-one as dermal penetration enhancers: side chain length variation and molecular modeling. - J. Control. Release, 73, 183-196, 2001. Percutaneous penetration enhancers, Smith E.W., Maibach H.I. Ed., Taylor & Francis, Boca Raton, London, New York, 2006. Harada S., Takahashi Y., Nakagawa H., Yamashita F., Hashida
20. 21. 22.
23. 24. 25. 26. 27. 28.
29.
30.
M. - Effect of vehicle properties on skin penetration of emedastine. - Biol. Pharm. Bull., 23, 1224-1228, 2000. Smith J.C., Irwin W.J. - Ionisation and the effect of absorption enhancers on transport of salicylic acid through silastic rubber and human skin. - Int. J. Pharm., 210, 69-82, 2000. Trotta M., Pattarino F., Gasco M.R. - Influence of counter ions on the skin permeation of methotrexate from water-oil microemulsions. - Pharm. Acta Helv., 71, 135-140, 1996. Sarveiya V., Templeton J.F., Benson H.A.E. - Effect of lipophilic counter-ions on membrane diffusion of benzydamine. - Eur. J. Pharm. Sci., 26, 39-46, 2005. Tantishaiyakul V., Phadoongsombut N., Wongpuwarak W., Thungtiwachgul J., Faroongsarng D., Wiwattanawongsa K., Rojanasakul Y. - ATR-FTIR characterization of transport properties of benzoic acid ion-pairs in silicone membranes. - Int. J. Pharm., 283, 111-116, 2004. Fang L., Numajiri S., Kobayashi D., Morimoto Y. - The use of complexation with alkanolamines to facilitate skin permeation of mefenamic acid. - Int. J. Pharm., 262, 13-22, 2003. Ratajczak H., Sobczyk L. - Dipole moments of hydrogen-bonded complexes and proton-transfer effect. - J. Chem. Phys., 50, 556-557, 1969. Jain A.K., Narishetty S.T.K., Panchagnula R. - Transdermal drug delivery of imipramine hydrochloride. I. Effect of terpenes. - J. Control. Release, 79, 93-101, 2002. Narishetty S.T.K., Panchagnula R. - Transdermal delivery of zidovudine: effect of terpenes and their mechanism of action. - J. Control. Release, 95, 367-379, 2004. Potts R.O., Guy R.H. - A predictive algorithm for skin permeability: the effects of molecular size and hydrogen bond activity. - Pharm. Res., 12, 1628-1633, 1995. Megrab N.A., Williams A.C., Barry B.W. - Oestradiol permeation across human skin, silastic and snake skin membranes: the effects of ethanol: water co-solvent system. - Int. J. Pharm., 116, 101-112, 1995. Femenía-Font A., Balaguer-Fern´andez C., Merino V., Rodilla V., López-Castellano A. - Effect of chemical enhancers on the in vitro percutaneous absorption of sumatriptan succinate. - Eur. J. Pharm. Biopharm., 61, 50-55, 2005. Wenkers B.P., Lippold B.C. - Skin penetration of nonsteroidal anti-inflammatory drugs out of a lipophilic vehicle: influence of the viable epidermis. - J. Pharm. Sci., 88, 1326-1331, 1999.
Acknowledgments The authors are grateful to Professor Yasunori Morimoto (Faculty of Pharmaceutical Sciences, Josai University, Japan) for his kind gift of two-chamber diffusion cells and a synchronous motor.
Manuscript Received 13 February 2008, accepted for publication 22 April 2008.
284
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang*, X.C. Niu, Y.X. Jiang, Z.G. He Department of Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning, 110016, China *Correspondence: [email protected] The purpose of the present study was to investigate the effect of ion-pairing on the permeation of glibenclamide through rat skin. Diethylamine, triethylamine, ethanolamine, diethanolamine and triethanolamine were used as counter-ions. The steady-state flux of glibenclamide markedly increased in the presence of counter-ions (P < 0.05). These results suggest that it is possible to enhance the permeation of glibenclamide by using an ion-pair approach. Moreover, the extent of enhancement possibly depends on the alkalinity, molecular weight, structure of the counter-ions and the dielectric constant of the donor solution. The influence of penetration enhancers on the permeation of glibenclamide was also determined. All the penetration enhancers studied increased the flux of glibenclamide compared with the control. Among these enhancers, menthol and N-methyl2-pyrrolidone showed the greatest enhancing activity. Key words: Glibenclamide – Ion-pairing – Penetration enhancers – Percutaneous absorption.
Glibenclamide is a potent, second-generation oral sulfonylurea hypoglycemic agent widely used in the treatment of non insulindependent diabetes. It lowers blood glucose levels by stimulating depolarization of pancreatic beta cells, therefore inducing the release of endogenous insulin [1]. However, many sulfonylureas have been associated with severe and sometimes fatal hypoglycemia and gastric disturbances like nausea, vomiting, heartburn, anorexia and increased appetite after oral therapy [2]. Since sulfonylureas are usually taken for a long period, the compliance of the patients is very important [3]. Due to the side effects after oral administration, the development of new preparations of glibenclamide is of significant importance. The transdermal route would be an attractive alternative for systemic delivery of glibenclamide, because it offers several advantages over conventional routes: avoidance of first-pass metabolism, the production of relatively constant drug plasma levels, a concurrent reduction in side effects, relative ease of drug input termination, and improved patient compliance. Nevertheless, the barrier function of the skin in protecting the body from physical and chemical attack makes delivery of the required drug dose through the skin and to the target organ difficult. A number of potential methods to enhance skin transport of drugs have been proposed, e.g. penetration enhancers, ion pair formation, prodrug design, iontophoresis, phonophoresis, and electroporation and some of them, including penetration enhancers and iontophoresis, have already been applied to the skin transport of glibenclamide [4-6]. Ion-pairs are defined as neutral species formed only by electrostatic attraction between oppositely charged ions [7], which are sufficiently lipophilic to dissolve in a lipoidal medium, such as the stratum corneum. The formation of ion-pairs to increase the skin penetration of drugs has been reported [8-10]. For example, Megwa et al. showed that secondary, tertiary and quaternary amines increased the in vitro permeation of salicylate across the human epidermis [11, 12]. Fang et al. found that the skin permeation of mefenamic acid increased in the presence of diethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine and propanolamine [13]. Sarveiya et al. reported increased penetration of ibuprofen through a polydimethylsiloxane membrane following ion-pair formation with alkylamines [14]. However, no information is available on the skin transport of glibenclamide following ion-pair formation. The objective of this study was to investigate the effect of ionpairing on the permeation of glibenclamide. In addition, the use of
penetration enhancers is a long-standing and widely used approach to increase transdermal and topical delivery [15]. In order to compare the permeation-enhancing effect of counter-ions with that of penetration enhancers, the influence of penetration enhancers on the permeation of glibenclamide was also evaluated.
I. Materials and methods 1. Materials
Glibenclamide (GLB) was purchased from Shandong Boshan Pharmaceuticals Co., Ltd. (Shandong, China). Diethylamine (DEA), triethylamine (TEA), ethanolamine (EtA), diethanolamine (DEtA), triethanolamine (TEtA), oleic acid and polyethylene glycol 400 (PEG400) were all supplied by Tianjin Bodi Chemicals Co., Ltd. (Tianjin, China). Methanol was of HPLC grade and purchased from Shandong Yuwang Chemicals Co., Ltd. (Shandong, China). Ethanol (EtOH), isopropyl myristate (IPM), N-methyl-2-pyrrolidone (NMP), azone and menthol were obtained from Tianjin Baishi Chemicals Co., Ltd. (Tianjin, China), China National Medicines Co., Ltd. (Shanghai, China), Beijing Chemical Industry (Beijing, China), Tianmen Kejie Pharmaceuticals Co., Ltd. (Hubei, China) and Chengdu Kelong Chemical Industry (Sichuan, China), respectively. All other chemicals and solvents were of analytical grade.
2. Preparation of skin samples
Male Wistar rats weighing 180-220 g (6-8 weeks old) used in all experiments were supplied by the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). The experiments were performed in accordance with the guidelines for animal use in the Life Science Research Center of Shenyang Pharmaceutical University. The rats were anesthetized with 20% urethane and then the hair from their abdomen was removed using animal hair clippers (model 900, TGC, Japan). Full thickness abdominal skin was harvested immediately after sacrificing the animals. The subcutaneous tissue and fat were carefully removed with surgical scissors, then the skin was washed with normal saline, wrapped in plastic pockets and stored at -20°C until use (within 1 week). The skin membrane was checked to ensure that no obvious defects were present.
3. In vitro permeation studies
The in vitro skin permeation experiments were carried out using
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Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
a side-by-side (2-chamber) diffusion cell having a volume of 2.5 mL and an effective diffusion area of 0.95 cm2. A piece of full-thickness rat skin was mounted between the two half cells and fastened with a spring clamp. The dermis side of the skin was in contact with the receiver compartment and the stratum corneum with the donor compartment. IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w) were used as donor solutions. The receiver compartment was filled with 2.5 mL pH 7.4 phosphate buffer containing 40% PEG 400 and the donor compartment with the drug suspension (with and without 5% enhancers or counter-ions at the same molar concentration as GLB). During all the experiments, excess drug was maintained in the donor compartment. Both compartments were then stirred at 600 rpm with a star-head bar driven by a synchronous motor and maintained at 32°C via water flow through a water jacket surrounding the cell. Samples (each 2 mL) were withdrawn from the receiver solution every 2 h over a period of 10 h and replaced with an equal volume of fresh receiver solution to maintain sink conditions. The experiments were performed at least in triplicate. The concentration of drug in the samples was analyzed, and the cumulative amount was plotted against time. The pseudo steady-state flux and lag time were determined as the slope of the linear portion of the plot and the intercept obtained by extrapolation of the linear portion to the time axis, respectively, and the permeability coefficient (P) was calculated by dividing the flux by the drug concentration in the donor phase. The enhancement ratio (ER) was calculated by dividing the cumulative amount of GLB permeated after 10 h (Q10) obtained with the strategies applied (penetration enhancers, counter-ions) by that of the control [16]. Statistical analysis was performed with Student’s t-test and a p-value of 0.05 or less was considered significant.
ben was used as the internal standard. The detector wavelength was 230 nm and the injection volume was 20 µL. The retention times of propylparaben and GLB were 5.2 and 8.5 min, respectively.
II. Results and discussion 1. Effect of ion-pairing on the permeation of GLB
In order to determine whether the polarity of the vehicle could influence the permeation of GLB in the presence of counter-ions through rat skin, permeation experiments were performed using IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w) as donor solutions. The effect of counter-ions on the permeation of GLB through rat skin from vehicle 1 (IPM: EtOH, 3:7) and 2 (IPM: EtOH, 7:3) is shown in Figure 1. The corresponding permeation parameters are presented in Table II. As seen in Table II, GLB was poorly soluble in both vehicles, and the addition of all counter-ions, except TetA, increased the solubility significantly (P < 0.05). For both vehicles, the steady-state flux of GLB in the presence of counter-ions was significantly higher (P < 0.05) than the control. The GLB flux observed with vehicle 1 follows the order: DEA ≈ TEA > DEtA > EtA > TEtA > control. DEA increased the Q10 by 7.24-fold, while TEA, DEtA, EtA and TEtA increased it by a factor of 7.19, 5.39, 2.90 and 2.41, respectively. There was no statistically significant difference between the enhancement produced by DEA and TEA. The flux observed with vehicle 2 was in the order of: EtA > DEA > TEA > DEtA > TEtA > control. EtA increased the Q10 by 41.58-fold, while DEA, TEA, DEtA and TEtA increased it by a factor of 28.87, 23.01, 12.15 and 5.44, respectively. No significant difference (P < 0.05) was observed between the Q10 of DEA and TEA. The formation of an ion-pair between GLB and counter-ions may be responsible for the enhanced skin permeation of GLB. In addition, the enhancing effect of all counter ions, except TEtA, can be attributed in part to the increased solubility of GLB in the donor phase. This may be explained by the simple equation for steady-state flux (Equation 1). When we plot the cumulative mass of diffusant, m, passing per unit area through a membrane, at long times the graph approaches linearity and its slope yields the steady flux, dm/ dt, as in the following equation [17]:
4. Analytical method
The concentration of GLB was determined by HPLC. The HPLC system consisted of a pump (HITACHI L-7100), a UV-Vis Detector (L-7420), a column temperature controller (HT-220A, Tianjin, China), a data station (T-2000L Tianmei Techcom, China), and a 5-µm ODS column (200 mm × 4.6 mm, DIKMA Technologies). The mobile phase was methanol - water (73:27, v/v) adjusted to pH 3.5 with phosphoric acid, and this was delivered at a flow rate of 1.0 mL/min. Propylpara-
(dm/dt) = (DC0K/b)
where C0 represents the constant donor drug concentration; K, the partition coefficient of solute between membrane and bathing solution; D, the diffusion coefficient; and b, the membrane thickness. From Equation 1, we can conclude that the flux may be increased by increasing the solubility of drug in the donor solution. Bjerrum’s equation [9], which describes a critical separation distance for the formation of an ion-pair, highlights the importance of the dielectric constant (e): a solvent with a high dielectric constant such as water (e = 78.5) is unfavorable for ion-pair formation, while the interaction becomes increasingly important in solvents with e < 40
Table I - Composition of the donor solutions. Vehicle
IPM (g)
EtOH (g)
GLB (mol)
Counter ions (mol)
Enhancers (g)
1
30 30 30
70 70 70
0.0081 0.0081 0.0081
0.0081 -
5
2
70 70 70
30 30 30
0.0081 0.0081 0.0081
0.0081 -
5
Eq. 1
Table II - Permeation parameters of GLB in the presence of various counter ions through rat skin from IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w) (mean ± SE of three experiments). Vehicle
Counter ion
Solubility (mg/mL)
Flux (µg/cm2/h)
Lag time (h)
ER
P x 103 (cm/h)
IPM:EtOH (3:7, w/w)
control DEA TEA EtA DEtA TEtA
1.17 2.84 4.46 12.00 6.98 0.32
9.64 ± 0.42 61.00 ± 9.13 59.35 ± 9.13 29.42 ± 2.81 51.80 ± 4.62 25.67 ± 4.34
3.73 ± 0.16 2.64 ± 0.98 2.65 ± 0.68 4.00 ± 0.20 3.74 ± 0.37 4.23 ± 0.48
7.24 ± 0.92 7.19 ± 1.34 2.90 ± 0.20 5.39 ± 0.72 2.41 ± 0.32
8.24 ± 0.36 21.49 ± 3.22 13.32 ± 2.05 2.45 ± 0.23 7.42 ± 0.66 79.95 ± 13.53
IPM:EtOH (7:3, w/w)
control DEA TEA EtA DEtA TEtA
0.77 1.11 2.56 3.50 5.36 0.68
10.09 ± 1.03 280.08 ± 39.71 272.72 ± 54.94 507.64 ± 28.46 136.75 ± 9.97 57.78 ± 4.57
3.92 ± 0.23 3.53 ± 0.38 4.95 ± 0.44 5.03 ± 0.18 4.67 ± 0.26 4.14 ± 0.50
28.87 ± 3.15 23.01 ± 5.38 41.58 ± 2.72 12.15 ± 1.03 5.44 ± 0.22
13.07 ± 1.34 252.55 ± 35.80 106.62 ± 21.48 145.00 ± 8.13 25.49 ± 1.86 84.74 ± 6.70
280
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
favorable for ion-pair formation. This suggests that the ion-pair skin transport of GLB follows Bjerrum’s equation. An increase in the lag time was observed when the concentration of IPM increased from 30 to 70%, indicating a certain reduction in the diffusion ability of GLB. This also suggests that EtOH is more potent in increasing the diffusion ability of GLB than IPM. Our results are in accordance with another report, where the diffusion ability (diffusion parameter) of emedastine decreased as the concentration of IPM increased from 27 to 63% [18]. In addition, the permeability coefficient of GLB increased markedly as the concentration of IPM increased from 30 to 70%. The permeability coefficient is equal to the product of the partition coefficient and the diffusivity of the drug in the membrane divided by the thickness of the membrane (assumed to be relatively constant) [23]. The increase in the permeability coefficient may therefore be due to the increase in the partition coefficient. The physicochemical properties of various counter-ions are summarized in Table III. It was observed that some properties of counterions could affect their permeation-enhancing effect significantly. The cumulative amount of GLB was found to increase as a function of the pKa value of the counter-ion except for EtA (Figure 2). A high pKa
(A) IPM:EtOH (3:7, w/w)
600
Cumulative amount permeated (?g/cm≤)
500
400
300
200
100
0 0
2
4
6
8
10
Time (h)
(B) IPM:EtOH (7:3, w/w)
3000
Table III - Physicochemical properties of various counter ions (Data were obtained from the SRC PhysProp Database).
2500
2000
1500
1000
Counter ion
pKa
log KO/W
Molecular weight
DEA TEA EtA DEtA TEtA
11.1 10.8 9.5 8.96 7.76
0.58 1.45 -1.31 -1.43 -1.00
73.14 101.19 61.08 105.14 149.19
(A) IPM:EtOH (3:7, w/w)
500 600
0
y = 103.76x - 566.02 2
R = 0.9196
500
0
2
4
6
8
10 Q (µg/cm2)
Cumulative amount permeated (?g/cm≤)
J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
Time (h)
Figure 1 - The effect of various counter-ions on the permeation of GLB through rat skin from IPM:EtOH (3:7, w/w) (A) and IPM:EtOH (7:3, w/w) (B). Each point represents the mean ± S.E. of three experiments. Key: (◊) control; (u) TEtA; (D) EtA; (s) DEtA; (p) DEA; (n) TEA.
400 300 200 100
[7]. A lipophilic system consisting of IPM and EtOH (3:7 or 7:3) was employed in this study. The dielectric constant of IPM and EtOH is 3.31 and 24.13, respectively [18]. To determine the significance of ion-pairing on the permeation of ionic drugs, phosphate buffer [8, 9], McIlvaine’s buffer [19], water-propylene glycol (70:30, w/w) [20], EtOH-pH 6.4 buffer solution (1:2, v/v) [10], propylene glycol (PG) [14, 21, 22] and EtOH-PG (2:1, v/v) [11, 12] have been used as donor solutions. PG is a solvent with a relatively low dielectric constant (e = 32.1) [22]. Compared with these donor solutions, the dielectric constant of the IPM/EtOH system is the lowest. Therefore, the IPM/ EtOH system is suitable for evaluating the effect of ion-pairing on the percutaneous absorption of ionic drugs. As shown in Table II, the flux of GLB in the presence of counter-ions increased significantly (P < 0.05) as the concentration of IPM increased from 30% to 70%. The dielectric constants of IPM:EtOH (27:73, v/v) and IPM:EtOH (63:37, v/v) are 18.36 and 9.13, respectively [18]. Since the relative percentage of IPM and EtOH in vehicle 1 and IPM:EtOH (27:73, v/v) is similar, the dielectric constant of vehicle 1 is similar to that of IPM:EtOH (27:73, v/v). Likewise, the dielectric constant of vehicle 2 is comparable to that of IPM:EtOH (63:37, v/v). The dielectric constant of vehicle 2 was lower than that of vehicle 1. The flux of GLB in the presence of counter-ions increased as the dielectric constant of the vehicle decreased, indicating that a vehicle with a low dielectric constant is
7
8
9
10
11
12
p Ka
(B) IPM:EtOH (7:3, w/w) 2000
y = 409.13x - 2873.1 2
Q (µg/cm2)
R = 0.9752 1500
1000
500
0 7
8
9
10
11
12
p Ka
Figure 2 - Relationship between the cumulative amount (Q) of GLB in the presence of counter-ions, except EtA, from IPM:EtOH (3:7, w/w) (A) and IPM:EtOH (7:3, w/w) (B) and the pKa of the counter-ions. Each point represents the mean of three experiments. 281
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Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
generally results in a high cumulative amount. The present results indicate that the enhancing effect of counter-ions is related to their alkalinity. The model of Huyskens and Zeegers-Huyskens predicts that a difference of 2.46-5.8 orders of magnitude between the acid dissociation constants of the base (pKa for DEA, TEA, EtA, DEtA, TEtA are 11.1, 10.8, 9.5, 8.96 and 7.76, respectively) and the acid (GLB, pKa = 5.3) leads to an almost complete shift of the proton-transfer equilibrium of the O-H···N ↔ O-···H-N+ system [24]. The larger the difference between the pKa of the base and acid, the larger the attractive force between the base and acid. In general, some structure activity relationships were apparent from our results in that the counter-ions with a hydroxyl functional group were less potent enhancers than those without a hydroxyl. Moreover, for vehicle 2, the flux of GLB decreased as the number of hydroxyls of the alkanolamine increased, indicating that the hydroxyl group has a negative effect on the permeation of GLB. It has been reported that oxygen-containing monoterpenes may preferentially form hydrogen bonds with ceramide head groups of stratum corneum [25, 26]. Likewise, alkanolamine with a hydroxyl group may also form hydrogen bonds with ceramide head groups. Potts and Guy found that hydrogen bonding ability had a negative effect on drug transport across the skin [27]. Therefore, the hydroxyl group may inhibit the permeation of ion pairs by forming hydrogen bonds with ceramide head groups. In addition, for vehicle 2, the cumulative amount of GLB increased with a decrease in the counter-ion molecular volume, expressed as a molecular weight (for many compounds, the molecular weight is often a reasonable approximation of the molecular volume) (Figure 3). For example, EtA, with the highest cumulative amount, has the lowest molecular weight. In contrast, TEtA, with the lowest cumulative amount, has a relatively high molecular weight. As the molecular weight of the counter-ions decreases, the cumulative amount of GLB increases to give an inverse relationship between the cumulative amount and the molecular weight. The importance of the molecular size of the counter-ions for the penetration of cephalexin through rat skin has been emphasized by Hatanaka et al. [9]. Megwa et al. also observed a close dependence of the flux of salicylate on the molecular size of the amine counter-ions [11]. Ion-pairs with small counter-ions are believed to have a small volume and, thus, may easily pass through the skin. However, for vehicle 1, there is no linear relationship between the cumulative amount of GLB and the molecular weight of counter-ions. This may be due to the fact that vehicle 1 with a higher EtOH concentration has a greater solvent drag effect of EtOH which probably obscures the smaller size effect. The flux of GLB does not correlate with the lipophilicity of the counter-ions expressed as logKO/W. For vehicle 2, the highest flux was measured with EtA. The highest ability to partition into n-octanol was measured with TEA but
2. Effect of penetration enhancers on the permeation of GLB
The effect of penetration enhancers on the permeation of GLB through rat skin from vehicle 1 and 2 is shown in Figure 4. The corresponding permeation parameters are summarized in Table IV. As can be seen from Table IV, the solubility of GLB in both vehicles increased significantly (P < 0.05) when penetration enhancers were used. For vehicle 1, only oleic acid did not produce a significant increase in the flux of GLB (P < 0.05). In all the other cases, the flux
Cumulative amount permeated (µg/cm2)
100
50
0
2
4
6
8
10
Time (h) 250
2
Log Q (µg/cm2)
150
0
y = -0.0099x + 4.011 R = 0.9363
3.5
(A) IPM:EtOH (3:7, w/w)
200
Cumulative amount permeated (µg/cm2)
4
the flux was 1.9-fold lower than that observed with EtA. Although the partition coefficient (logKO/W) of EtA is similar to that of DEtA, the flux of GLB in the presence of EtA is 3.7-fold higher than that in the presence of the latter. Similar results have been reported for lignocaine [8]. Among the counter-ions examined, TEtA exhibited the lowest penetration- enhancing effect in the present study. There may be several explanations for this. The solubility of GLB was the lowest in the presence of TEtA. In addition, the formation of ion pairs is only possible if the ions approach each other and reach a critical separation distance [7]. However, the tertiary alkanol group of TEtA may make it difficult for the nitrogen atom of TEtA to approach the GLB molecule.
3
2.5
(B) IPM:EtOH (7:3, w/w)
200
150
100
50
0 2
0 40
60
80
100
120
140
2
4
6
8
10
160
Time (h) MW
Figure 4 - In vitro permeation profiles of GLB in the presence of various penetration enhancers through rat skin from IPM:EtOH (3:7, w/w) (A) and IPM:EtOH (7:3, w/w) (B). Each point represents the mean ± SE of three experiments. Key: (◊) control; (u) oleic acid; (D) azone; (s) NMP; (p) menthol.
Figure 3 - Relationship between the logarithm of the cumulative amount (Q) of GLB in the presence of counter-ions from IPM:EtOH (7:3, w/w) and the molecular weight (MW) of the counter-ions. Each point represents the mean of three experiments. 282
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
of GLB showed statistically significant differences with respect to the control (P < 0.05). Menthol provided the highest increase in flux, followed by Azone, NMP and oleic acid. Similar to the flux, the highest increase in Q10 was observed with menthol, which increased the Q10 by 2.24-fold, followed by NMP (1.45-fold), azone (1.45-fold), and oleic acid (1.08-fold). Azone and menthol significantly shortened the lag time of GLB compared with the control (P < 0.05). For vehicle 2, all the penetration enhancers tested significantly enhanced the flux of GLB in comparison to the control (P < 0.05). NMP provided the highest increase in flux, followed by menthol, azone and oleic acid. However, the highest increase in Q10 was observed with menthol, which increased the Q10 by 3.06-fold, followed by NMP (2.89-fold), oleic acid (1.86-fold), and azone (1.77-fold). There was a significant decrease in the lag time (P < 0.05) of GLB by menthol when compared with the control. The permeation-enhancing effect of enhancers may be attributed partially to the increased solubility of GLB in the donor phase. As shown in Table III, the increase in EtOH concentration in vehicle from 30 to 70% (w/w) results in a slight decrease in flux. Megrab et al. reported that the decreased permeation of a lipophilic drug (estradiol) from vehicles with higher EtOH concentrations was due to dehydration of the stratum corneum [28]. The decrease in the permeability of GLB through rat skin may also be due to dehydration of the lamellar layers of the stratum corneum. The solubility of GLB in vehicle 1 was significantly higher (P < 0.05) than that in vehicle 2, indicating that EtOH has a greater ability to dissolve GLB compared with IPM. Considering the results obtained with vehicle 2, it seems that there is an inverse relationship between the lipophilicity of enhancers expressed as logKO/W (n-octanol/water partition coefficient) and their enhancing activity (Figure 5). The highest enhancing effect was obtained with NMP and menthol, compounds with logKO/W values of -0.38 and 3.40, whereas more lipophilic compounds, such as Azone (logKO/W = 6.28) and oleic acid (logKO/W = 7.73), had lower capacity to increase the flux of GLB. However, Femenía-Font et al. found that there is an optimum lipophilicity of the promoting agent as far as enhancing activity is concerned [29]. This discrepancy was due to the different vehicles, barriers and drugs employed in the two studies. As logKO/W increases, the enhancer becomes more like the vehicle (the enhancer should be more soluble in the vehicle). For enhancers with a low logKO/W, the interaction between enhancers and the stratum corneum will probably be strong because the vehicle has a weak affinity for enhancers. Conversely, for enhancers with a high logKO/W, the vehicle has a strong affinity for enhancers and, therefore, the interaction will be weak. Thus, enhancers with a high lipophilicity exhibit low enhancing activity. In contrast, the enhancing effect of enhancers with a low lipophilicity should be high. However, for vehicle 1, we could not obtain a good linear correlation of the lipophilicity of enhancers with their enhancing activity. The lipophilicity of vehicle 1 is lower than that of vehicle 2. For enhancers with a low logKO/W, the interaction between enhancers and the stratum corneum will probably be weaker because vehicle 1 has a stronger affinity for enhancers compared to
J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
y = -1.5989x + 29.287
35
2
R = 0.9413
30
Flux (µg/cm2/h)
25 20 15 10 5 0 -1
0
1
2
3
4
5
6
7
8
Log K o/w
Figure 5 - Relationship between the fluxes of GLB in the presence of various penetration enhancers from IPM:EtOH (7:3, w/w) and the noctanol/water partition coefficient (logKO/W) of the penetration enhancers. Each point represents the mean of three experiments.
vehicle 2. Conversely, for enhancers with a high logKO/W, the vehicle has a weaker affinity for enhancers and, therefore, the interaction will be stronger compared to vehicle 2. Thus, the lipophilicity dependency of vehicle 1 is much less than that of vehicle 2. The enhancing effect of the counter ions is greater than that of penetration enhancers. Some researchers reported that the viable epidermis, not the stratum corneum, is the rate-limiting barrier for the transport of very lipophilic chemicals [18, 23, 30]. Since GLB is a lipophilic drug, the viable epidermis is the rate-limiting step in skin penetration rather than the stratum corneum. Penetration enhancers increase the transdermal drug penetration by altering the barrier function of the stratum corneum. Counter-ions may decrease the lipophilicity of GLB by forming an ion-pair and hence may improve the partition of GLB into the viable epidermis. The penetration of GLB is determined by the viable epidermis and the counter-ions are therefore more effective than penetration enhancers for increasing the permeation of GLB through rat skin. * The results of this study suggest that it is possible to increase the permeation of GLB by using an ion-pair approach. Counter-ions are more efficient than penetration enhancers for increasing the permeation of GLB through rat skin. Moreover, the degree of enhancement possibly depends on the alkalinity, molecular weight, structure of the counter-ions and the dielectric constant of the donor solution. Among the penetration enhancers examined, menthol and NMP showed the greatest enhancement activity, indicating that they could be promising penetration enhancers for transdermal delivery systems for GLB.
Table IV - Permeation parameters of GLB in the presence of various penetration enhancers through rat skin from IPM:EtOH (3:7, w/w) and IPM:EtOH (7:3, w/w). Vehicle
Enhancers
Solubility (mg/mL)
Flux (µg/cm2/h)
Lag time (h)
ER
P x 103 (cm/h)
IPM:EtOH (3:7, w/w)
control NMP azone oleic acid menthol
1.17 2.06 1.62 1.61 1.42
9.64 ± 0.42 12.79 ± 1.63 13.46 ± 0.16 10.15 ± 2.10 18.86 ± 3.04
3.73 ± 0.16 3.13 ± 0.51 3.45 ± 0.11 3.45 ± 0.40 2.73 ± 0.07
1.45 ± 0.21 1.45 ± 0.03 1.08 ± 0.19 2.24 ± 0.35
8.24 ± 0.36 6.21 ± 0.79 8.32 ± 0.10 6.32 ± 1.31 13.32 ± 2.15
IPM:EtOH (7:3, w/w)
control NMP azone oleic acid menthol
0.77 1.69 1.03 0.90 1.09
10.09 ± 1.03 29.03 ± 1.75 18.09 ± 3.23 16.94 ± 3.35 25.86 ± 5.40
3.92 ± 0.23 3.86 ± 0.19 4.00 ± 0.40 3.06 ± 0.53 2.56 ± 0.43
2.89 ± 0.08 1.77 ± 0.31 1.86 ± 0.26 3.06 ± 0.48
13.07 ± 1.34 17.21 ± 1.04 17.56 ± 3.14 18.86 ± 3.73 23.68 ± 4.94
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J. DRUG DEL. SCI. TECH., 18 (4) 279-284 2008
Effect of ion-pairing on the permeation of glibenclamide through rat skin R. Ma, L. Fang, X.C. Niu, Y.X. Jiang, Z.G. He
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Acknowledgments The authors are grateful to Professor Yasunori Morimoto (Faculty of Pharmaceutical Sciences, Josai University, Japan) for his kind gift of two-chamber diffusion cells and a synchronous motor.
Manuscript Received 13 February 2008, accepted for publication 22 April 2008.
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