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Effect of counter-ions and penetration enhancers on the skin permeation of flurbiprofen
Effect of Counter-Ions and Penetration Enhancers on the Skin Permeation of Flurbiprofen XU MA,1 LIANG FANG,1 JIANPENG GUO,2 NANXI ZHAO,1 ZHONGGUI HE1 1
Department of Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, China 2
College of Pharmacy, Yanbian University, 1829 Juzi Street, Yanji, Jilin 133000, China
Received 24 April 2009; revised 17 August 2009; accepted 23 August 2009 Published online 5 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21953
ABSTRACT: The aim of this work was to investigate the percutaneous absorption of flurbiprofen (FP) using counter-ions as enhancers as well as to compare their enhancing activity with penetration enhancers in vitro. The in vitro permeation studies of FP were performed in isopropyl myristate (IPM) solution by two-chamber diffusion cells, using rat abdominal skin as a model. Among the penetration enhancers examined, including the cosolvents of propylene glycol and ethanol (EtOH), oleic acid, menthol, laurocapram, sorbitan monooleate, and N-methyl-2-pyrrolidone (NMP), 10% (w/w) EtOH and NMP exhibited the most potent solubilization and enhancing effects of FP from IPM, with a flux of (372.60 45.12) mg/cm2/h and (474.21 46.64) mg/cm2/h, respectively. Ten percent (w/w) EtOH/IPM binary system was used to investigate the effect of the counter-ions, namely diethylamine (DEA), triethylamine (TEA), ethanolamine (EtA), diethanolamine (DEtA), triethanolamine (TEtA), and N-(20 -hydroxyethanol)-piperdine (HEPP). The cumulative amounts were markedly increased in the presence of the counter-ions, and the highest flux of (1297.53 121.81) mg/cm2/h was obtained by DEA. This was related to the decreased lipophilicity and different physicochemical properties of the ion-pairs. In particular, we proved the formation of an FP/amine ion-pair in solution by 1H-NMR. The results suggest that the counter-ions are more efficient than penetration enhancers. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1826–1837, 2010
Keywords: flurbiprofen; transdermal; penetration enhancer; organic amine; ionpairing; percutaneous absorption; solubility; in vitro; HPLC; 1H-NMR
INTRODUCTION Over the past three decades, the skin has become an important portal for drug delivery for topical, regional, and systemic actions. Dermal and transdermal drug delivery is often limited by the poor permeability of the skin to drugs, which precludes their crossing the skin at therapeutic
Correspondence to: Liang Fang 23986330; Fax: 86-24-23986330; E-mail: [email protected])
(Telephone:
86-24-
Journal of Pharmaceutical Sciences, Vol. 99, 1826–1837 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
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rates. Flurbiprofen (FP) is a nonsteroidal antiinflammatory drug (NSAID), one of the most potent inhibitors of platelet aggregation currently available, is used to treat gout, osteoarthritis, rheumatoid arthritis, and sunburn.1,2 United States FDA has approved FP tablets, which often have a disadvantage of gastrointestinal side effects and a frequent dosing.3 FP patches are available in Japan and Korea et al., but the product is large in size and the permeation needs to be further enhanced yet. Thus, an effective and convenient method to develop a safe and low-cost transdermal drug delivery system for FP is in need. Nevertheless, one particular problem is that
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FP was one of the least-permeable drugs across skin among a series of lipophilic drugs.4 It was previously reported that full-thickness skin represents an artificially high barrier towards the percutaneous absorption of hydrophobic drugs. Based on the two-layer skin model, this is because drugs with very low water solubility do not partition freely from the stratum corneum (SC) into the aqueous environment of the viable epidermis (ED).5,6 This has been attributed to the dermal tissues which, being essentially an aqueous barrier, inhibit the partitioning of hydrophobic substance from the lipophilic SC. Likewise, FP is a lipophilic drug with a log Ko/w of 3.86, indicating that besides the SC another key rate-limiting barrier for the percutaneous absorption of FP is supposed to be the ED. Alternative strategies to overcome the problems arising from poor permeability and solubility need to be developed. To achieve this goal, various strategies were investigated. Fang et al.7,8 examined the effect of penetration enhancers on FP. Charoo et al.9 studied the permeability of FP by using tulsi (Ocimum sanctum) and turpentine oil. Swart et al.10 synthesis and examined the increase in skin absorption of the derivatives of NSAIDs including FP and Jain et al.11 prepared the solid lipid nanoparticles of FP in order to enhance the permeability of FP. However, these methods were not very effective in improving the hydrophilicity of FP and then enhancing the permeability of FP, since another rate-limiting barrier effect of the ED has been ignored. The formation of ion-pairs are reported to increase the skin penetration of drugs12–14 and improve the lipophilicity of the drugs. Early studies on ion-pair transport focused on absorption from the gastrointestinal tract. Wilson and Wiseman were among the first to test the ion-pair hypothesis for the lipophilization of the ionic drug tropsium.15 They reported an enhanced transfer rate across everted intestine using alkylsulphonates as counter-ions. Gasco and colleagues reported and increase in the bioavailability of propranol in the presence of taurodeoxycholate.16 The concept of forming ion-pairs to increase the skin permeability of hydrophilic drugs has also been reported.17–20 In recent years, the ion-pair effect on lipophilic drug have been focus point. For example, Megwa et al. showed that secondary, tertiary and quaternary amines increased the in vitro permeation of salicylate across the human ED.21,22 Fang et al. found that the skin permeation of mefenamic acid increased DOI 10.1002/jps
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in the presence of diethylamine (DEA), triethylamine (TEA), ethanolamine (EtA), diethanolamine (DEtA), triethanolamine (TEtA), and N-(20 -hydroxyethanol)-piperdine (HEPP).23 Sarveiya and colleagues reported increased penetration of ibuprofen through a polydimethylsiloxane membrane following ion-pair formation with alkyl amines.24 Wang et al. increased scutellarin permeability by forming ion-pair with DEA, TEA, EtA, DEtA, and TEtA, and proved the presence of ion-pair formation by 1H-NMR.25 In recent years, Maitre confirmed the formation of FP with hydroxypropyl-b-cyclodextrin (HP-b-CD) and three EtAs by DSC, and studied the binary FP–EtA complex with HP-b-CD transdermal system.26 Nevertheless, there is still no published systemic evaluation of ion-pair formation of FP. The goal of this study was to evaluate the percutaneous absorption of FP using counter-ions as enhancers as well as to compare their enhancing activity with chemical enhancers in vitro. The cosolvents (propylene glycol—PG and EtOH), and other penetration enhancers (oleic acid—OA, menthol, laurocapram, sorbitan monooleate, Nmethyl-2-pyrrolidone—NMP) were examined. Six organic amines (DEA, TEA, EtA, DEtA, TEtA, and HEPP) were used as counter-ions. This research was performed in IPM solution using a two-layer diffusion model. The effect of 1H-NMR spectroscopy was used to identify an ion-pair formation between FP and the counter-ions.
MATERIALS AND METHODS Materials FP was purchased from Shanghai Haiqu Chemical Co., Ltd (Shanghai, China); EtA, DEtA, TEtA, DEA, TEA, HEPP were purchased from the Yuwang Pharmaceutical Co., Ltd (Shandong, China); PG, ethanol (EtOH), oleic acid (OA), Azone (AZ), Span-80 (SP) were obtained from the Bodi Drug Manufacturing Co., Ltd (Tianjin, China); isopropyl myristate (IPM), NMP, L-menthol (MT) were supplied by China National Medicines Co., Ltd (Shanghai, China). Methanol was of HPLC grade and was obtained from the Yuwang Pharmaceutical Co., Ltd. All other chemicals were of the highest reagent grade available. A phosphate-buffered saline (PBS) at pH 7.4 (add 1.36 g KH2PO4 and 79 mL of 0.1 M NaOH to make 200 mL, pH 7.4, phosphate buffer) was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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prepared according to the Chinese Pharmacopoeia (2005). The water used was deionized and distilled in an all-glass still, and it will be referred to as distilled water. A 0.1 mm cellulose nitrate 0.45mm pore size membrane was obtained from FLMTechnology Development Co., Ltd. (Tianjin, China). Drug Analysis The quantitative determination of FP was performed by HPLC with a methanol solution of propylparaben as internal standard and with reference to a calibration curve. A Hitachi instrument (pump L-2130, UV–Vis detector L2420, T2000L workstation) and a Hypersil ODS 5 mm 200 mm 4.6 mm column (Dalian Elite Analytical Instruments Co., Ltd, Dalian, China) were used. The mobile phase consisted of methanol and 0.5% glacial acetic acid in distilled water (75:25, v/v). The column was maintained at 408C and the flow rate was 1 mL/min while the UV detector was set at 245 nm. Retention times in this assay were found to be 4.69 and 5.97 min for internal standard and FP, respectively. The Hypersil ODS analytical column and the mobile phase used for the assay provided a well-defined separation between the drug and internal standard. There were no interferences from the endogenous components. Determination of Drug Solubility and the Calculation of the Solubility Parameter The solubility of drugs in IPM and the EtOH/IPM system, with or without incorporating various adjuvants, were determined at 328C. Excessive FP was added to the vehicle, with and without incorporating equimolar amount of organic amines. An excess of each drug was dispersed into 2 mL solution in a sealed glass vial. Each vial was shaken in a water bath for 48 h until equilibrium was achieved, and then 1 mL was transferred to a polypropylene micro-vial and centrifuged. The concentration of FP and its complexes in the samples was assayed by HPLC after appropriate dilution with methanol. The Hansen solubility parameters of the compounds were calculated from the chemical structures using the approaches of Hoftyzer/Van Krevelen.27 The calculation of the solubility parameter was based on the average molecular weight. The units of the solubility parameters are (J cm3)1/2.28 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
Skin Sample Preparation Wistar male 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 published by the Life Science Research Center of Shenyang Pharmaceutical University. The rats were anesthetized with urethane (20%, w/w, i.p.) and the abdomen carefully shaved with a razor after removal of hair by electric clippers (model 900, TGC, Japan). About 5 cm2 (circle of 2.5 cm diameter) of fullthickness skin was excised from the shaved site. The fat and subdermal tissues were removed with surgical scissors. The skin was washed with normal saline and kept frozen at 208C. The skin was checked to ensure that no obvious defects were present prior to the experiments.
Permeation Experiments Permeation experiments were conducted at 328C in two-chamber diffusion cells (with an effective diffusion area 0.95 cm2 and receiver volume of 2.5 mL).23 After removal of hair and subcutaneous fat, the rat abdominal skin membrane was mounted on a two-chamber diffusion cell with the epidermal side facing the donor cell. The donor cell was filled with a 2.5 mL suspension of FP, about twice the solubility in each solvent system. The excess solid of FP in the suspension ensured drug saturation conditions in the donor phase throughout the experiment (clearly, the drug solubility value will be different, depending on the vehicle composition), and the solubility of FP in donor phase was sufficient for the permeation. The receptor cell was filled with 2.5 mL PBS (pH 7.4) and at predetermined time intervals, 2.0 mL of receptor solution was sampled for analysis and replaced with the same volume of fresh solution to maintain sink conditions. The drug concentration was determined by HPLC with reference to a calibration curve.
Data Analysis All experiments were replicated at least four times. The amount of each drug permeating through the skin during a sampling interval was calculated based on the measured receptorphase concentration and volume. The 8-h DOI 10.1002/jps
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cumulative amount (Q8 h) of drug permeating per unit area versus time was plotted. All data were calculated and presented as mean SE. The skin flux was determined from Fick’s law of diffusion: JðfluxÞ ¼
dQr Adt
(1)
where J is the steady-state skin flux in mg/cm2/h, dQr is the change in quantity of the drug passing through the skin into the receptor compartment in mg, A is the active diffusion area in cm2, and dt is the change in time. The flux was calculated from the slope of the linear portion of the profiles. The x-intercept of the linear portion of the plot was the lag-time. The permeability coefficient ( P) was obtained by dividing the flux by the initial drug concentration in the donor phase. The penetration-enhancing effect of the adjuvant was calculated in terms of the enhancement ratio (ER), using the following equation: ER ¼
Q8 h ; withenhancers Q8 h ; control
RESULTS AND DISCUSSION The Effect of PG and EtOH on the Permeation of FP
(2)
Statistical analysis was carried out using analysis of variance (ANOVA) with the help of the SPSS program. The level of significance was taken as p < 0.05.
1H-NMR Spectrometry The 1H-NMR measurements were carried out at 600 MHz on a Bruker ARX 600 spectrometer (ARX-600, Bruker, Zurich, Switzerland). FP (10 mg) with various equimolar organic amines were added to separate NMR tubes and DMSO was added as solvent, including pure FP as a control formulation. The protons of FP were assigned as illustrated in Figure 1. Chemical shifts for proton resonance were reported in ppm relative to TMS.
Figure 1. Structures of FP, with proton assignment illustrated for FP. DOI 10.1002/jps
Figure 2. Effect of PG and EtOH on the permeation of FP through rat abdominal skin. (Each point represents the mean SE of three to five experiments.)
The effect of the cosolvents (PG and EtOH) in different ratios on the permeation of FP through rat skin was examined. The permeation profiles obtained are shown in Figure 2. The corresponding permeation parameters are presented in Table 1. As seen from Table 1, the 5% PG/IPM, 5%, 10%, and 15% EtOH/IPM were all increased the cumulative amount permeated (Q8 h) of FP ( p < 0.05), while the 10% PG/IPM had a significance of p ¼ 0.052. The ER of Q8 h with PG was 1.28 in 10% PG/IPM and 2.36 in 5% PG/IPM, while the ER of FP with EtOH was 5.00, 5.51, 5.57 in 5% EtOH/IPM, 10% EtOH/IPM, 15% EtOH/IPM, respectively. The rank order of the solubility of FP in the EtOH/IPM binary solvent system was in accordance with the Q8 h. As is shown in Table 1, the most suitable ratio was obtained for 10% EtOH, with the highest flux of 372.60 mg/cm2/h. While the final amount of FP permeating from pure IPM and 5%, 10% PG/IPM and 5%, 15% EtOH/IPM was 146.69, 76.49, 352.09, 331.70 mg/ cm2/h, respectively. The skin permeation of FP from PG/IPM system was found to be concentration independent, and the largest permeation ratio of PG was 5% in IPM system. Moreover, EtOH is a more effective solvent and penetration enhancer than PG for improving the solubility of FP and the permeation. The resultant increase in the steady state flux of FP can be attributed in part to the increased solubility of FP in the donor phase, and the solvent drag effect of EtOH might JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Table 1. Permeation Parameters of FP Through Rat Skin Using an Identical Receiver Phase (PBS) and Donor Phases Consisting of IPM; 5% PG/IPM; 10% PG/IPM; 5% EtOH/IPM; 10% EtOH/IPM; 15% EtOH/IPM (w/w) (n ¼ 3–5) Vehicle (w/w) IPM 10% PG/IPM 5% PG/IPM 5% EtOH/IPM 10% EtOH/IPM 15% EtOH/IPM
Q8 h (mg/cm2)
Flux (mg/cm2/h)
TLag (h)
Solubility (mg/mL)
p (cm/h 103)
ER
445.62 46.72 568.42 20.55 1050.42 90.23 2227.26 67.17 2453.38 157.47 2482.33 173.29
69.01 7.45 76.49 3.70 146.69 10.62 352.09 17.69 372.6 45.12 331.70 13.5
1.53 0.11 0.52 0.1 0.94 0.33 1.66 0.16 1.04 0.17 0.50 0.25
24.66 28.12 26.49 53.21 58.18 76.05
2.80 2.72 5.54 6.61 6.40 4.36
— 1.28 2.35 5.00 5.51 5.57
partly contribute to the enhanced skin permeation of the drug, indicating that other factors such as partitioning into the skin from the EtOH/IPM system are involved,29 in addition to greater membrane fluidity and pore formation associated directly with the increasing EtOH content.30
The Effect of Different Penetration Enhancers on the Permeation of FP A lipohilic vehicle of IPM was investigated in this study, since little systematic information has been published on the effect of lipophilic vehicles on FP. Based on a two-layer diffusion model, Fang et al. have investigated a series of NSAIDs with different log Ko/w (0.50–4.88) across hairless rat intact and stripped skin from IPM, indicated that IPM partition into the relatively highly ordered region of the lipid bilayer induces disorder and increase the fluidity in this region. The disorder that is induced in this region leads to an increased effective free volume and increased effective diffusivity in this region.29 It has been shown theoretically that ‘‘hydrophilic’’ drugs will be enhanced most by agents which have a positive impact on the SC diffusion process. Conversely, ‘‘lipophilic’’ drugs will be transported more efficiently if the enhancer can, in some way, facilitate the SC-viable tissue partitioning step.31 Some reports29,32 have shown that when using the lipophilic vehicle IPM, the SC did not seem to offer significant resistance although the viable ED is the rate-limiting barrier for the transport of relatively hydrophobic drugs. FP is lipophilic and practically insoluble in water (i.e., 0.14 mM33), indicating that IPM as a vehicle is fit to use in research. The effectiveness of different penetration enhancers on the permeation of FP through rat skin from IPM is shown in Figure 3. The corresponding parameters are given in Table 2. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
As seen from Table 2, the steady state flux and solubility of FP were all increased than control when penetration enhancers were used. AZ, MT, OA, SP, and NMP increased the Q8 h ( p < 0.05) with an ER of 2.26, 2.29, 2.49, 3.21, and 6.67, respectively. Among these penetration enhancers, NMP exerted the greatest solubilization on drug in the donor phase, in which the solubility of FP increased from 24.66 to 81.72 mg/mL and this is supported by previous studies.34 Akhter and Barry reported that NMP enhanced the skin permeation of FP and ibuprofen applied as solid drug film because the dissolution step was removed, and thermodynamic activity of the drug remaining in the solution increased after rapid permeation of NMP and NMP changed the diffusion resistance of the skin.35 In addition, the structure of NMP shows that there is a lactam group in the molecule, which is expected to exhibit H-bonding with FP.36 In our study, NMP showed the greatest solubilization among those penetration enhancers, which can be partially interpreted by H-bonding with the drug. However, OA, SP,
Figure 3. Effect of penetration enhancers on the permeation of FP through rat abdominal skin. (Each point represents the mean SE of three to five experiments.) DOI 10.1002/jps
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Table 2. Permeation Parameters of FP Through Rat Skin After Adding Different Penetration Enhancers Using an Identical Receiver Phase (PBS) and Donor Phases Consisting of IPM (n ¼ 3–5) Vehicle (w/w) IPM 5% AZ 5% MT 5% OA 5% SP 5% NMP
Q8 h (mg/cm2)
Flux (mg/cm2/h)
TLag (h)
Solubility (mg/mL)
p (cm/h 103)
ER
445.62 46.72 1006.52 71.61 1018.86 76.50 1108.14 225.64 1429.27 123.45 2973.37 244.45
69.01 7.45 145.15 7.55 162.54 8.62 153.60 28.12 248.47 20.83 474.21 46.64
1.53 0.11 1.15 0.11 1.80 0.19 0.89 0.34 2.29 0.25 1.61 0.31
24.66 44.86 31.33 28.79 29.47 81.72
2.80 3.24 5.19 5.34 8.43 5.80
— 2.26 2.29 2.49 3.21 6.67
and MT may preferentially formed intermolecular and intramolecular hydrogen bonds with autochthonous molecules, resulting in a decrease in the number of binding sites of hydrogen bond with drug.25
The Effect of Various Organic Amines on the Permeation of FP Since a lipophilic multicomponent system consisting of TEtA-10% EtOH/IPM system has been shown to selectively enhance the skin permeation of acidic drugs,23 and 10% EtOH/IPM system was suitable for the permeation of FP according to previous study. We investigated the effects of six organic amines (DEA, TEA, HEPP, EtA, DEtA, and TEtA) on the permeation of FP. The permeation profiles of FP in 10% EtOH/IPM binary solvent system obtained are shown in Figure 4.
Figure 4. Effect of various organic amines on the permeation of FP through rat abdominal skin. (Each point represents the mean SE of three to five experiments.) DOI 10.1002/jps
The corresponding permeation parameters are presented in Table 3. As illustrated in Figure 4 and Table 3, all the counter-ions employed in present study had an obviously positive effect on FP permeation ( p < 0.05). The Q8 h of FP increased to (9228.53 172.26) mg/cm2 in DEA, (7194.82 852.19) mg/cm2 in TEA, (6390.69 534.44) mg/ cm2 in EtA, (5566.21 194.95) mg/cm2 in TEtA, (4951.23 280.81) mg/cm2 in DEtA, (4562.55 730.59) mg/cm2 in HEPP, respectively. And the solubility of FP was increased markedly after adding organic amines, increasing the solubility of FP from 24.66 mg/mL in IPM to 118.81– 462.48 mg/mL in FP/amine-10% EtOH/IPM system. Bjerrum’s equation,37 which describes a critical separation distance for the formation of an ionpair, highlights the importance of the dielectric constant (e): a solvent with a high dielectric constant such as water (e ¼ 78.5) is unfavorable for ion-pair formation, while the interaction becomes increasingly important in solvents with e < 40.38 The donor vehicle consisted of 90% (w/w) IPM and 10% (w/w) EtOH, and the dielectric constant of the two solvents was 3.31 and 24.13, respectively;39 the calculated dielectric constant of the donor vehicle was 5.39.40–42 Thus, the 10% (w/w) EtOH/IPM employed in this research is suitable for evaluation of the effect of ionpairing on the percutaneous absorption of ionic drugs. 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. It is reported that the flux of drug decreased as the number of hydroxyls of the alkanolamine increased, indicating that the hydroxyl group has a negative effect on the permeation of drug. It has been reported that oxygen-containing monoterpenes may JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Table 3. Permeation Parameters of FP Through Rat Skin in the Presence of Various Counter-Ions Which Molar Quantities Were Equal to FP Dissolved in 10% EtOH/IPM(w/w) (n ¼ 3–5) Vehicle IPM 10% EtOH/IPM HEPP DEtA TEtA EtA TEA DEA
Q8 h (mg/cm2)
Flux (mg/cm2/h)
TLag (h)
Solubility (mg/mL)
p (cm/h 103)
ER
445.62 46.72 2453.38 157.47 4562.55 730.59 4951.23 280.81 5566.21 194.95 6390.69 534.44 7194.82 852.19 9228.53 172.26
69.01 7.45 372.6 45.12 751.54 169.82 496.16 12.28 752.45 10.73 759.19 120.86 1231.38 60.30 1297.53 121.81
1.53 0.11 1.04 0.17 1.67 0.54 0 0.76 0.20 0.46 0.46 2.11 0.60 0.89 0.70
24.66 58.18 152.01 118.81 248.28 306.50 462.48 257.81
2.80 6.40 4.94 4.18 3.03 2.47 2.66 5.03
— 5.51 10.24 11.11 12.49 14.34 16.15 20.71
preferentially form hydrogen with ceramide head groups of SC.43,44 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.45 Therefore, the hydroxyl group may inhibit the permeation of ion-pairs by forming hydrogen bonds with ceramide head groups. The physicochemical properties of various organic amines are summarized in Table 4. It was observed that some properties of organic amines could also affect their permeation-enhancing effect. The flux of FP was found to increase as a function of pKa value of the counter-ion except TEtA (Fig. 5), indicating 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, HEPP, TEtA are 11.1, 10.8, 9.5, 8.96, 9.04, and 7.76, respectively) and the acid (FP, pKa ¼ 4.2)46 leads to an almost complete shift of the protontransfer equilibrium of the O–H N $ O– H–
Nþ system.47 The larger the difference between the pKa of the amine and acid, the larger the attractive force between the amine and acid, and may lead to a high flux. TEtA has a low pKa but a relative high flux, this maybe because TEtA has the most hydroxyl groups among these organic amines, possibly there is a strong H-bonding action with FP just as the effect of NMP, which mainly lead to increasing the solubility and the permeation of FP. The Hansen solubility parameters of the counter-ions and FP were shown in Table 4 and calculated based on the methods of Hoftyzer/Van Krevelen and Hoy.27 Compounds with similar values for d are likely to be miscible. This is because the energy of mixing released by interactions within the components is balanced by the energy released by interaction between the components.48 Greenhalgh et al. classified excipients based on the difference between the solubility parameters of the excipients and drugs (Dd). The authors demonstrated that compounds with a Dd < 7.0 Mpa1/2 were likely to be miscible. When the Dd > 10.0 Mpa1/2 the compounds were likely to be immiscible. From the results
Table 4. Physicochemical Properties of the Organic Amines and FP
HEPP DEtA TEtA EtA TEA DEA FP
Formula
MW
Log Ko/w
pKa
da
C7H15NO NH(CH2 CH2OH)2 HOCH2CH2N(CH2 CH2OH)2 H2NCH2 CH2OH N(CH2CH3)3 NH(CH2CH3)2 C15H13FO2
129.20 105.14 149.19 61.08 101.19 73.14 244.26
0.96 1.43 1.00 1.31 1.45 0.58 3.86b
9.04 8.96 7.76 9.5 10.8 11.1 4.2c
24.24 28.87 30.42 27.28 16.10 15.83 22.05
Data were obtained from SRC PhysProp Database. a d Solubility parameter. b Morimoto et al.4 c Tang-Liu et al.46 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Figure 5. Relationship between the flux of FP in the presence of counter-ions except TEtA from 10% EtOH/ IPM (w/w) and the pKa of the counter-ions. (Each point represents the mean of three to five experiments.)
summarized in Table 4 the amines (d for DEA, TEA, EtA, TEtA, DEtA, HEPP, are 15.83, 16.10, 27.28, 30.42, 28.87, 24.24, respectively) and FP (d ¼ 22.05) in this study should show some miscibility with a range of Dd from 2.19 to 8.37. An inverse relationship between the flux and d of the counter-ions is shown in Figure 6. Then based on the good miscibility between the drug and the counter-ions, the counter-ion with a decrease in solubility parameter (an increase in lipophilicity) exhibited an increase in the flux of FP. Such behavior could be elucidated by ion-pair formation and the partition between the SC and the ED.29 The excised rat skin contains the ED (divided into the SC and the viable ED) and the dermis. The viable ED and dermis (ED) are similar and regarded as an aqueous protein gel.49 Drugs penetrate the skin according to the following
Figure 6. Relationship between the flux of FP in the presence of six counter-ions from 10% EtOH/IPM (w/w) and the d (solubility parameter) of the counter-ions. (Each point represents the mean of three to five experiments.) DOI 10.1002/jps
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process, during which drugs partition between the EI system and the SC firstly, and then diffuse through the SC. Subsequently, they partition between the SC and the ED, and finally diffuse through the ED. Once they reach the dermis, drugs are absorbed by the blood capillaries within minutes. Lipophilic drugs pass through the SC, which is essentially a lipid layer, and then they must transfer directly into the aqueous medium— ED, otherwise they will remain in the SC. According to the theory, the main rate-limiting step for lipophilic drug is the partition from the SC to the ED. So the affinity for the ED, in other words the hydrophilicity, is an important factor to lipophilic drug. This has also been confirmed by the study of Jiang et al.50 Therefore, improving the hydrophilicity of lipophilic drug may facilitate the partition between the SC to the ED. Addition of counter-ions is reported to be an effective method to obtain a balanced hydrophilicity– lipophilicity for both hydrophilic and lipophilic drugs. In case of a hydrophilic drug, such as 5aminolevulinic acid51 and methotrexate,52 a lipophilic counter-ion can be used to enhance the lipophilicity of the drug and hence promote the SC diffusion process. In contrast a hydrophilic counter-ion should be used for lipophilic drugs, such as benzydamine53 and mefenamic acid.29 Therefore, ‘‘lipophilic’’ drugs will be transported more efficiently if the counter-ion can, in some way, facilitate the SC-viable tissue partitioning step.31 It agreed with the present study that addition of hydrophilic counter-ions improved the hydrophilicity of FP, and then facilitates the SC– ED partitioning step. After adding the hydrophilic counter-ions, the ‘‘ion-pair’’ as a unit showed the similar characteristic as hydrophilic drugs, since the ‘‘ion-pair’’ had an increase in affinity (lipophilicity) to SC exhibited an increase in the flux of FP. As indicated above, the enhancing effect of the counter-ions is greater than that of penetration enhancers. Since FP is a lipophilic drug, the viable ED, not the SC, is the rate-limiting step in skin penetration.29,54,55 Penetration enhancers may mainly increase the transdermal drug penetration by altering the barrier function of the SC. Counter-ions may decrease the lipophilicity of FP by forming an ion-pair and hence may promote the key step of the partition of FP into the ED. The penetration of FP is determined by the viable ED, so the counter-ions are more effective than penetration enhancers for increasing the permeation of FP through rat skin. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Table 5.
a b c d e f g h i j
1
H NMR Chemical Shifts of FP and its Ion-Pairs for Proton on Carbon
FP
DEA
TEA
EtA
TEtA
DEtA
HEPP
7.22 7.24 7.41 7.47 7.49 7.54 3.78 1.41 Overlap Overlap
7.19 7.20 7.37 7.45 7.47 7.52 3.52 (þ0.26) 1.33 (þ0.08) Overlap Overlap
7.21 7.22 7.40 7.45 7.47 7.53 3.66 (þ0.12) 1.37 (þ0.04) Overlap Overlap
7.17 7.19 7.37 7.45 7.47 7.52 3.40 (þ0.38) 1.31 (þ0.10) Overlap Overlap
7.22 7.24 7.41 7.47 7.49 7.54 3.75 (þ0.03) 1.40 (þ0.01) Overlap Overlap
7.19 7.20 7.39 7.45 7.47 7.52 3.52 (þ0.26) 1.34 (þ0.07) Overlap Overlap
7.21 7.23 7.40 7.46 7.48 7.53 3.68 (þ0.10) 1.38 (þ0.03) Overlap Overlap
NMR Discussion The goal of the 1H-NMR measurement was to obtain evidence for the presence of ion-pair formation between FP and respective counterions from the chemical shift changes to protons near the carboxyl group, as ion-pair formation is an indicator of increased permeability of the counter-ions through skin. Table 5 shows the modulation of the FP assigned protons signals in the different formulations. The changes of chemical shifts of the ionpairs relative to FP are obviously indicated in Figure 7. It is clear that addition of equimolar counter-ions to FP resulted in no significant shifts of aromatic protons having shifts of only 0.01– 0.05 ppm. However, the (g) and (h) protons have obviously changed in chemical shifts, which have been the focus of this study. Assignment (g) and (h) represent a single proton and a methyl group (three equivalent protons), respectively, which attached to the carboxyl group. The proton (h) shifted upfield by 0.01–0.08 ppm, while the proton (g) signal was considerably shifted upfield by 0.03–0.38 ppm. The high shifts exhibited by protons g and h suggest that these groups were
particularly influenced by bonding between counter-ions and FP, which is indicative of ion-pair formation. It could be elucidated that to FP molecule, the carboxyl group plays a role of electrophilic action on proton (g) and (h), which leads to a decrease of electron atmosphere density and a deshielding. After application of counterions, the carboxyl group of FP salified with respective bases, which decreased the electrophilic action, thus, the proton (g) and (h) shifted upfield. Similar bonding involving carboxylic acids and amine groups has been reported by Hosono et al.56; Sanders and Hunter57; Beten et al.58; Servet;59 and Sarveiya et al.24
CONCLUSION This work evaluated the enhancing effect of counter-ions on FP as well as penetration enhancers and proved the formation of ion-pairs. We can conclude that counter-ions are more efficient than penetration enhancers with a combination of 10% EtOH/IPM system. Of all the amines studied, DEA produced the greatest
Figure 7. The chemical shifts of the assigned protons of FP. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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enhancement of FP, which may be influenced by pKa, its structure and hydrophilicity. Among the penetration enhancers investigated, NMP was found to be the most promising chemical enhancer, attributed to the altering barrier function of SC and its solubilization on FP. We will synthesis various FP–amine complexes, and optimize the formulation of these complexes in the form of patch in further research.
10.
11.
12.
ACKNOWLEDGMENTS The authors are grateful to Professor Yasunori Morimoto (Faculty of Pharmaceutical Sciences, Josai University, Japan) for his kind gift of twochamber diffusion cells and a synchronous motor.
REFERENCES 1. Lee DJ, Burt CT, Koch RL. 1992. Percutaneous absorption of flurbiprofen in the hairless rat measured in vivo using 19F magnetic resonance spectroscopy. J Invest Dermatol 99:431–434. 2. Poul J, West J, Buchanan N, Grahame R. 1993. Local action transcutaneous flurbiprofen in the treatment of soft tissue rheumatism. Br J Pharmatol 32:1000–1003. 3. Heyneman CA, Lawless-Liday C, Wall GC. 2000. Oral versus topical NSAID in rheumatic disease: A comparison. Drugs 60:555–574. 4. Morimoto Y, Hatanaka T, Sugibayashi K, Omiya H. 1992. Prediction of skin permeability of drugs: Comparison of human and hairless rat skin. J Pharm Pharmacol 44:634–639. 5. Bronaugh RL, Stewart RF. 1984. Method for in vitro percutaneous absorption studies III: Hydrophobic compounds. J Pharm Sci 73:1255–1258. 6. Bronaugh RL, Stewart RF. 1986. Method for in vitro percutaneous absorption studies VI: Preparation of the barrier layer. J Pharm Sci 75:487– 491. 7. Fang JY, Hwang TL, Fang CL, Chiu HC. 2003. In vitro and in vivo evaluations of the efficacy and safety of skin permeation enhancers using flurbiprofen as a model drug. Int J Pharm 255: 153–166. 8. Fang JY, Chiu HC, Wu JT, Chiang YR, Hsuc SH., 2004. Fatty acids in Botryococcus braunii accelerate topical delivery of flurbiprofen into and across skin. Int J Pharm 276:163–173. 9. Charoo NA, Shamsher AAA, Kohli K, Pillai K, Rahman Z. 2008. Improvement in bioavailability of
DOI 10.1002/jps
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
1835
transdermally applied flurbiprofen using tulsi (Ocimum sanctum) and turpentine oil. Colloids Surf B Biointerfaces 65:300–307. Swart H, Breytenbach JC, Hadgraft J, Plessis J. 2005. Synthesis and transdermal penetration of NSAID glycoside esters. Int J Pharm 301:71– 79. Jain SK, Chourasia MK, Masuriha R, Soni V, Jain A, Jain NK, Gupta Y. 2005. Solid lipid nanoparticles bearing flurbiprofen for transdermal delivery. Drug Deliv 12:207–215. Valenta C, Siman U, Kratzel M, Hadgraft J. 2000. The dermal delivery of lignocaine: Influence of ion pairing. Int J Pharm 20:77–85. Hatanaka T, Kamon T, Morigaki S, Katayama K, Koizumi T. 2000. Ion pair skin transport of a zwitterionic drug, cephalexin. J Control Release 66:63– 71. Trotta M, Ugazio E, Peira E, Pulitano C. 2003. Influence of ion pairing on topical delivery of retinoic acid from microemulsions. J Control Release 86:315–321. Wilson TH, Wiseman G. 1954. The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J Physiol 123:116–125. Gasco M, Eandi M. 1984. The influence of bile salts on the absorption in vitro and in vivo of propranolol. Pharm Biomed Anal 2:424–428. Hadgraft J, Walters KA, Wotton PK. 1985. Facilitated transport of sodium salicylate across an artificial lipid membrane by Azone. J Pharm Pharmacol 37:725–727. Hadgraft J, Walters KA, Wotton PK. 1986. Facilitated percutaneous absorption: A comparison and evaluation of two in vitro models. Int J Pharm 32:257–263. Young C-S, Shi C-K, Lee M-H, Kim S-K. 1988. Effect of sodium salicylate on in vitro percutaneous penetration of isopropamide iodide through mouse skin. Int J Pharm 45:59–64. Pedersen M. 1990. Synergistic action of clotrimazole and certain anionic surfactants may be due to ion pair formation. Acta Pharm Nord 2:367– 370. Megwa SA, Cross SE, Benson HAE, Roberts MS. 2000. Ion-pair formation as a strategy to enhance topical delivery of salicylic acid. J Pharm Pharmacol 52:919–928. Megwa SA, Cross SE, Whitehouse MW, Benson HAE, Roberts MS. 2000. Effect of ion pairing with alkylamines on the in-vitro dermal penetration and local tissue disposition of salicylates. J Pharm Pharmacol 52:929–940. Fang L, Kobayashi Y, Numajiri S, Kobayashi D, Sugibayashi K, Morimoto Y. 2002. The enhancing effect of a triethanolamine-ethanol-isopropyl myr-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
1836
24.
25.
26.
27. 28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
MA ET AL.
istate mixed system on the skin permeation of acidic drugs. Biol Pharm Bull 25:1339–1344. Sarveiya V, Templeton JF, Benson HAE. 2004. Ionpairs of ibuprofen: Increased membrane diffusion. J Pharm Pharmacol 56:717–724. Wang ML, Fang L, Ren CS, Li T. 2008. Effect of ionpairing and enhancers on scutellarin skin permeability. J Pharm Pharmacol 60:429–435. Maitre MM, Longhi MR, Gladys GG. 2007. Ternary complexes of flurbiprofen with HP-b-CD and ethanolamines characterization and transdermal delivery. Drug Dev Ind Pharm 33:311–326. Krevelen V, Krevelen DW. 1990. Properties of Polymers. Amsterdam: Elsevier Science Publishing Co. Hancock BC, York P, Rowe RC. 1997. The use of solubility parameters in pharmaceutical solid dosage form design. Int J Pharm 148:1–21. Fang L, Numajiri S, Kobayashi D, Morimoto Y. 2003. The use of complexation with alkanolamines to facilitate skin permeation of mefenamic acid. Int J Pharm 262:13–22. McDaid DM, Deasy PB. 1996. Formulation development of a transdermal drug delivery system for amlodipine base. Int J Pharm 133:71–83. Hori M, Satoh S, Maibach HI, Guy RH. 1991. Enhancement of propranolol hydrochloride and diazepam skin absorption in vitro: Effect of enhancer lipophilicity. J Pharm Sci 80:32–35. Wenkers BP, Lippold BC. 1999. Skin penetration of non-steroidal anti-inflammatory drugs out of a lipophilic vehicle: Influence of the viable epidermis. J Pharm Sci 88:1326–1331. Tavornvipas S, Hirayama F, Arima H, Uekama K, Ishiguro T, Oka M, Hamayasu K, Hashimoto H. 2002. 6-O-a-(4-O-a-D-glucuronyl) -D-glucosyl-ßcyclodextrin: Solubilizing ability and some cellular effects. Int J Pharm 249:199–209. Sawa CL, Heng PW, Chin WW, Soo KC, Olivo M. 2006. Enhanced photodynamic activity of hypericin by penetration enhancer N-methyl pyrrolidone formulations in the chick chorioallantoic membrane model. Cancer Lett 238:104–110. Akhter S, Barry BW. 1985. Absorption through human skin of ibuprofen and flurbiprofen; effect of dose variation, deposited drug film, occlusion and the penetration enhancer N-methyl-2-pyrrolidone. J Pharm Pharmacol 37:27–37. Lee PJ, Ahmad N, Langer R, Mitragotri S, Prasad SV. 2006. Evaluation of chemical enhancers in the transdermal delivery of lidocaine. Int J Pharm 308: 33–39. Hatanaka T, Kamon T, Morigaki S, Katayama K, Koizumi T. 2000. Ion pair skin transport of a zwitterionic drug, cephalexin. J Control Release 66:63– 71. Kraus CA. 1956. The ion pair concept: Its evolution and some applications. J Phys Chem 60:129– 141.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
39. Harada S, Takahashi Y, Nakagawa H, Yanmashita F, Hashida MB. 2000. Effect of vehicle properties on skin penetration of emedastine. Pharm Bull 23: 1224–1228. 40. Prakongpan S, Nagai T. 1984. Solubility of acetaminophen in cosolvents. Chem Pharm Bull 32:340– 343. 41. Chien YW. 1984. Solubilization of metronidazole by watermiscible multi-cosolvents and water-soluble vitamins. J Parent Sci Technol 38:32–36. 42. Dumanovic D, Kosanovic DJ, Ardakovic D, Jovanovic J. 1992. Solubilization of ipronidazole by co-solvents. Pharmazie 47:603–607. 43. Jain AK, Narishetty STK, Panchagnula R. 2002. Transdermal drug delivery of imipramine hydrochloride. I. Effect of terpenes. J Control Release 79:93–101. 44. Narishetty STK, Panchagnula R. 2004. Transdermal delivery of zidovudine: Effect of terpenes and their mechanism of action. J Control Release 95: 367–379. 45. Potts RO, Guy RH. 1995. A predictive algorithm for skin permeability: The effects of molecular size and hydrogen bond activity. Pharm Res 12:1628–1633. 46. Tang-Liu DD, Liu S. 1987. Relationship between the ocular and systemic disposition of flurbiprofen: The effect of altered protein dynamics at steady state. J Pharm and Bio 15:387–397. 47. Ratajczak H, Sobczyk L. 1969. Dipole moments of hydrogen-bonded complexes and proton-transfer effect. J Chem Phys 50:556–557. 48. Greenhalgh D, Williams A, Timmins P, York P. 1999. Solubility parameters as predictors of miscibility in solid disperisons. J Pharm Sci 88:1182–1190. 49. Scheuplein RJ. 1976. Mechanism of percutaneous absorption II. Transient diffusion and the relative importance of various routes of skin permeation. J Invest Dermatol 48:79–88. 50. Jiang Y, Fang L, Niu X, Ma R, He Z. 2008. The effect of ion pairing on the skin permeation of amlodipine. Pharmazie 63:356–360. 51. Auner BG, Valenta C, Hadgraft J. 2003. Influence of lipophilic counter-ions in combination with phloretin and 6-ketocholestanol on the skin permeation of 5-aminolevulinic acid. Int J Pharm 255:109–116. 52. Trotta M, Pattarino F, Gasco MR. 1996. Influence of counter ions on the skin permeation of methotrexate from water-oil microemulsions. Pharm Acta Helv 71:135–140. 53. Sarveiya V, Templeton JF, Benson HAE. 2005. Effect of lipophilic counterions on membrane diffusion of benzydamine. Eur J Pharm Sci 26:39–46. 54. Wenkers BP, Lippold BC. 1999. Skin penetration of non-steroidal antiinflammatory drugs out of a lipophilic vehicle: Influence of the viable epidermis. J Pharm Sci 88:1326–1331. 55. Harada S, Takahashi Y, Nakagawa H, Yanmashita F, Hashida M. 2000. Effect of vehicle properties on
DOI 10.1002/jps
EFFECT OF COUNTER-IONS AND PENETRATION ENHANCERS
skin penetration of emedastine. Biol Pharm Bull 23:1224–1228. 56. Hosono T, Tsuchiya S, Matsumaru H. 1979. Molecular complex formation between N-vinyl-2-pyrrolidone and ajmaline. Chem Pharm Bull 27:58–64. 57. Sanders JKM, Hunter BK. 1989. Modern NMR Spectroscopy. Oxford: Oxford University Press. p. 215.
DOI 10.1002/jps
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58. Beten DB, Gelbcke M, Diallo B, Moes AJ. 1992. Interaction between dipyridamole and Eudragit, S. Int J Pharm 88:31–37. 59. Servet B, Nadir B, Rytting JH. 1996. Interaction of indomethacin with a new penetration enhancer, dodecyl 2-(N,N-dimethylamino)propionate (DDAIP): Its effect on transdermal delivery. Int J Pharm 127:245–253.
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Department of Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, China 2
College of Pharmacy, Yanbian University, 1829 Juzi Street, Yanji, Jilin 133000, China
Received 24 April 2009; revised 17 August 2009; accepted 23 August 2009 Published online 5 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21953
ABSTRACT: The aim of this work was to investigate the percutaneous absorption of flurbiprofen (FP) using counter-ions as enhancers as well as to compare their enhancing activity with penetration enhancers in vitro. The in vitro permeation studies of FP were performed in isopropyl myristate (IPM) solution by two-chamber diffusion cells, using rat abdominal skin as a model. Among the penetration enhancers examined, including the cosolvents of propylene glycol and ethanol (EtOH), oleic acid, menthol, laurocapram, sorbitan monooleate, and N-methyl-2-pyrrolidone (NMP), 10% (w/w) EtOH and NMP exhibited the most potent solubilization and enhancing effects of FP from IPM, with a flux of (372.60 45.12) mg/cm2/h and (474.21 46.64) mg/cm2/h, respectively. Ten percent (w/w) EtOH/IPM binary system was used to investigate the effect of the counter-ions, namely diethylamine (DEA), triethylamine (TEA), ethanolamine (EtA), diethanolamine (DEtA), triethanolamine (TEtA), and N-(20 -hydroxyethanol)-piperdine (HEPP). The cumulative amounts were markedly increased in the presence of the counter-ions, and the highest flux of (1297.53 121.81) mg/cm2/h was obtained by DEA. This was related to the decreased lipophilicity and different physicochemical properties of the ion-pairs. In particular, we proved the formation of an FP/amine ion-pair in solution by 1H-NMR. The results suggest that the counter-ions are more efficient than penetration enhancers. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1826–1837, 2010
Keywords: flurbiprofen; transdermal; penetration enhancer; organic amine; ionpairing; percutaneous absorption; solubility; in vitro; HPLC; 1H-NMR
INTRODUCTION Over the past three decades, the skin has become an important portal for drug delivery for topical, regional, and systemic actions. Dermal and transdermal drug delivery is often limited by the poor permeability of the skin to drugs, which precludes their crossing the skin at therapeutic
Correspondence to: Liang Fang 23986330; Fax: 86-24-23986330; E-mail: [email protected])
(Telephone:
86-24-
Journal of Pharmaceutical Sciences, Vol. 99, 1826–1837 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
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rates. Flurbiprofen (FP) is a nonsteroidal antiinflammatory drug (NSAID), one of the most potent inhibitors of platelet aggregation currently available, is used to treat gout, osteoarthritis, rheumatoid arthritis, and sunburn.1,2 United States FDA has approved FP tablets, which often have a disadvantage of gastrointestinal side effects and a frequent dosing.3 FP patches are available in Japan and Korea et al., but the product is large in size and the permeation needs to be further enhanced yet. Thus, an effective and convenient method to develop a safe and low-cost transdermal drug delivery system for FP is in need. Nevertheless, one particular problem is that
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FP was one of the least-permeable drugs across skin among a series of lipophilic drugs.4 It was previously reported that full-thickness skin represents an artificially high barrier towards the percutaneous absorption of hydrophobic drugs. Based on the two-layer skin model, this is because drugs with very low water solubility do not partition freely from the stratum corneum (SC) into the aqueous environment of the viable epidermis (ED).5,6 This has been attributed to the dermal tissues which, being essentially an aqueous barrier, inhibit the partitioning of hydrophobic substance from the lipophilic SC. Likewise, FP is a lipophilic drug with a log Ko/w of 3.86, indicating that besides the SC another key rate-limiting barrier for the percutaneous absorption of FP is supposed to be the ED. Alternative strategies to overcome the problems arising from poor permeability and solubility need to be developed. To achieve this goal, various strategies were investigated. Fang et al.7,8 examined the effect of penetration enhancers on FP. Charoo et al.9 studied the permeability of FP by using tulsi (Ocimum sanctum) and turpentine oil. Swart et al.10 synthesis and examined the increase in skin absorption of the derivatives of NSAIDs including FP and Jain et al.11 prepared the solid lipid nanoparticles of FP in order to enhance the permeability of FP. However, these methods were not very effective in improving the hydrophilicity of FP and then enhancing the permeability of FP, since another rate-limiting barrier effect of the ED has been ignored. The formation of ion-pairs are reported to increase the skin penetration of drugs12–14 and improve the lipophilicity of the drugs. Early studies on ion-pair transport focused on absorption from the gastrointestinal tract. Wilson and Wiseman were among the first to test the ion-pair hypothesis for the lipophilization of the ionic drug tropsium.15 They reported an enhanced transfer rate across everted intestine using alkylsulphonates as counter-ions. Gasco and colleagues reported and increase in the bioavailability of propranol in the presence of taurodeoxycholate.16 The concept of forming ion-pairs to increase the skin permeability of hydrophilic drugs has also been reported.17–20 In recent years, the ion-pair effect on lipophilic drug have been focus point. For example, Megwa et al. showed that secondary, tertiary and quaternary amines increased the in vitro permeation of salicylate across the human ED.21,22 Fang et al. found that the skin permeation of mefenamic acid increased DOI 10.1002/jps
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in the presence of diethylamine (DEA), triethylamine (TEA), ethanolamine (EtA), diethanolamine (DEtA), triethanolamine (TEtA), and N-(20 -hydroxyethanol)-piperdine (HEPP).23 Sarveiya and colleagues reported increased penetration of ibuprofen through a polydimethylsiloxane membrane following ion-pair formation with alkyl amines.24 Wang et al. increased scutellarin permeability by forming ion-pair with DEA, TEA, EtA, DEtA, and TEtA, and proved the presence of ion-pair formation by 1H-NMR.25 In recent years, Maitre confirmed the formation of FP with hydroxypropyl-b-cyclodextrin (HP-b-CD) and three EtAs by DSC, and studied the binary FP–EtA complex with HP-b-CD transdermal system.26 Nevertheless, there is still no published systemic evaluation of ion-pair formation of FP. The goal of this study was to evaluate the percutaneous absorption of FP using counter-ions as enhancers as well as to compare their enhancing activity with chemical enhancers in vitro. The cosolvents (propylene glycol—PG and EtOH), and other penetration enhancers (oleic acid—OA, menthol, laurocapram, sorbitan monooleate, Nmethyl-2-pyrrolidone—NMP) were examined. Six organic amines (DEA, TEA, EtA, DEtA, TEtA, and HEPP) were used as counter-ions. This research was performed in IPM solution using a two-layer diffusion model. The effect of 1H-NMR spectroscopy was used to identify an ion-pair formation between FP and the counter-ions.
MATERIALS AND METHODS Materials FP was purchased from Shanghai Haiqu Chemical Co., Ltd (Shanghai, China); EtA, DEtA, TEtA, DEA, TEA, HEPP were purchased from the Yuwang Pharmaceutical Co., Ltd (Shandong, China); PG, ethanol (EtOH), oleic acid (OA), Azone (AZ), Span-80 (SP) were obtained from the Bodi Drug Manufacturing Co., Ltd (Tianjin, China); isopropyl myristate (IPM), NMP, L-menthol (MT) were supplied by China National Medicines Co., Ltd (Shanghai, China). Methanol was of HPLC grade and was obtained from the Yuwang Pharmaceutical Co., Ltd. All other chemicals were of the highest reagent grade available. A phosphate-buffered saline (PBS) at pH 7.4 (add 1.36 g KH2PO4 and 79 mL of 0.1 M NaOH to make 200 mL, pH 7.4, phosphate buffer) was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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prepared according to the Chinese Pharmacopoeia (2005). The water used was deionized and distilled in an all-glass still, and it will be referred to as distilled water. A 0.1 mm cellulose nitrate 0.45mm pore size membrane was obtained from FLMTechnology Development Co., Ltd. (Tianjin, China). Drug Analysis The quantitative determination of FP was performed by HPLC with a methanol solution of propylparaben as internal standard and with reference to a calibration curve. A Hitachi instrument (pump L-2130, UV–Vis detector L2420, T2000L workstation) and a Hypersil ODS 5 mm 200 mm 4.6 mm column (Dalian Elite Analytical Instruments Co., Ltd, Dalian, China) were used. The mobile phase consisted of methanol and 0.5% glacial acetic acid in distilled water (75:25, v/v). The column was maintained at 408C and the flow rate was 1 mL/min while the UV detector was set at 245 nm. Retention times in this assay were found to be 4.69 and 5.97 min for internal standard and FP, respectively. The Hypersil ODS analytical column and the mobile phase used for the assay provided a well-defined separation between the drug and internal standard. There were no interferences from the endogenous components. Determination of Drug Solubility and the Calculation of the Solubility Parameter The solubility of drugs in IPM and the EtOH/IPM system, with or without incorporating various adjuvants, were determined at 328C. Excessive FP was added to the vehicle, with and without incorporating equimolar amount of organic amines. An excess of each drug was dispersed into 2 mL solution in a sealed glass vial. Each vial was shaken in a water bath for 48 h until equilibrium was achieved, and then 1 mL was transferred to a polypropylene micro-vial and centrifuged. The concentration of FP and its complexes in the samples was assayed by HPLC after appropriate dilution with methanol. The Hansen solubility parameters of the compounds were calculated from the chemical structures using the approaches of Hoftyzer/Van Krevelen.27 The calculation of the solubility parameter was based on the average molecular weight. The units of the solubility parameters are (J cm3)1/2.28 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
Skin Sample Preparation Wistar male 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 published by the Life Science Research Center of Shenyang Pharmaceutical University. The rats were anesthetized with urethane (20%, w/w, i.p.) and the abdomen carefully shaved with a razor after removal of hair by electric clippers (model 900, TGC, Japan). About 5 cm2 (circle of 2.5 cm diameter) of fullthickness skin was excised from the shaved site. The fat and subdermal tissues were removed with surgical scissors. The skin was washed with normal saline and kept frozen at 208C. The skin was checked to ensure that no obvious defects were present prior to the experiments.
Permeation Experiments Permeation experiments were conducted at 328C in two-chamber diffusion cells (with an effective diffusion area 0.95 cm2 and receiver volume of 2.5 mL).23 After removal of hair and subcutaneous fat, the rat abdominal skin membrane was mounted on a two-chamber diffusion cell with the epidermal side facing the donor cell. The donor cell was filled with a 2.5 mL suspension of FP, about twice the solubility in each solvent system. The excess solid of FP in the suspension ensured drug saturation conditions in the donor phase throughout the experiment (clearly, the drug solubility value will be different, depending on the vehicle composition), and the solubility of FP in donor phase was sufficient for the permeation. The receptor cell was filled with 2.5 mL PBS (pH 7.4) and at predetermined time intervals, 2.0 mL of receptor solution was sampled for analysis and replaced with the same volume of fresh solution to maintain sink conditions. The drug concentration was determined by HPLC with reference to a calibration curve.
Data Analysis All experiments were replicated at least four times. The amount of each drug permeating through the skin during a sampling interval was calculated based on the measured receptorphase concentration and volume. The 8-h DOI 10.1002/jps
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cumulative amount (Q8 h) of drug permeating per unit area versus time was plotted. All data were calculated and presented as mean SE. The skin flux was determined from Fick’s law of diffusion: JðfluxÞ ¼
dQr Adt
(1)
where J is the steady-state skin flux in mg/cm2/h, dQr is the change in quantity of the drug passing through the skin into the receptor compartment in mg, A is the active diffusion area in cm2, and dt is the change in time. The flux was calculated from the slope of the linear portion of the profiles. The x-intercept of the linear portion of the plot was the lag-time. The permeability coefficient ( P) was obtained by dividing the flux by the initial drug concentration in the donor phase. The penetration-enhancing effect of the adjuvant was calculated in terms of the enhancement ratio (ER), using the following equation: ER ¼
Q8 h ; withenhancers Q8 h ; control
RESULTS AND DISCUSSION The Effect of PG and EtOH on the Permeation of FP
(2)
Statistical analysis was carried out using analysis of variance (ANOVA) with the help of the SPSS program. The level of significance was taken as p < 0.05.
1H-NMR Spectrometry The 1H-NMR measurements were carried out at 600 MHz on a Bruker ARX 600 spectrometer (ARX-600, Bruker, Zurich, Switzerland). FP (10 mg) with various equimolar organic amines were added to separate NMR tubes and DMSO was added as solvent, including pure FP as a control formulation. The protons of FP were assigned as illustrated in Figure 1. Chemical shifts for proton resonance were reported in ppm relative to TMS.
Figure 1. Structures of FP, with proton assignment illustrated for FP. DOI 10.1002/jps
Figure 2. Effect of PG and EtOH on the permeation of FP through rat abdominal skin. (Each point represents the mean SE of three to five experiments.)
The effect of the cosolvents (PG and EtOH) in different ratios on the permeation of FP through rat skin was examined. The permeation profiles obtained are shown in Figure 2. The corresponding permeation parameters are presented in Table 1. As seen from Table 1, the 5% PG/IPM, 5%, 10%, and 15% EtOH/IPM were all increased the cumulative amount permeated (Q8 h) of FP ( p < 0.05), while the 10% PG/IPM had a significance of p ¼ 0.052. The ER of Q8 h with PG was 1.28 in 10% PG/IPM and 2.36 in 5% PG/IPM, while the ER of FP with EtOH was 5.00, 5.51, 5.57 in 5% EtOH/IPM, 10% EtOH/IPM, 15% EtOH/IPM, respectively. The rank order of the solubility of FP in the EtOH/IPM binary solvent system was in accordance with the Q8 h. As is shown in Table 1, the most suitable ratio was obtained for 10% EtOH, with the highest flux of 372.60 mg/cm2/h. While the final amount of FP permeating from pure IPM and 5%, 10% PG/IPM and 5%, 15% EtOH/IPM was 146.69, 76.49, 352.09, 331.70 mg/ cm2/h, respectively. The skin permeation of FP from PG/IPM system was found to be concentration independent, and the largest permeation ratio of PG was 5% in IPM system. Moreover, EtOH is a more effective solvent and penetration enhancer than PG for improving the solubility of FP and the permeation. The resultant increase in the steady state flux of FP can be attributed in part to the increased solubility of FP in the donor phase, and the solvent drag effect of EtOH might JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Table 1. Permeation Parameters of FP Through Rat Skin Using an Identical Receiver Phase (PBS) and Donor Phases Consisting of IPM; 5% PG/IPM; 10% PG/IPM; 5% EtOH/IPM; 10% EtOH/IPM; 15% EtOH/IPM (w/w) (n ¼ 3–5) Vehicle (w/w) IPM 10% PG/IPM 5% PG/IPM 5% EtOH/IPM 10% EtOH/IPM 15% EtOH/IPM
Q8 h (mg/cm2)
Flux (mg/cm2/h)
TLag (h)
Solubility (mg/mL)
p (cm/h 103)
ER
445.62 46.72 568.42 20.55 1050.42 90.23 2227.26 67.17 2453.38 157.47 2482.33 173.29
69.01 7.45 76.49 3.70 146.69 10.62 352.09 17.69 372.6 45.12 331.70 13.5
1.53 0.11 0.52 0.1 0.94 0.33 1.66 0.16 1.04 0.17 0.50 0.25
24.66 28.12 26.49 53.21 58.18 76.05
2.80 2.72 5.54 6.61 6.40 4.36
— 1.28 2.35 5.00 5.51 5.57
partly contribute to the enhanced skin permeation of the drug, indicating that other factors such as partitioning into the skin from the EtOH/IPM system are involved,29 in addition to greater membrane fluidity and pore formation associated directly with the increasing EtOH content.30
The Effect of Different Penetration Enhancers on the Permeation of FP A lipohilic vehicle of IPM was investigated in this study, since little systematic information has been published on the effect of lipophilic vehicles on FP. Based on a two-layer diffusion model, Fang et al. have investigated a series of NSAIDs with different log Ko/w (0.50–4.88) across hairless rat intact and stripped skin from IPM, indicated that IPM partition into the relatively highly ordered region of the lipid bilayer induces disorder and increase the fluidity in this region. The disorder that is induced in this region leads to an increased effective free volume and increased effective diffusivity in this region.29 It has been shown theoretically that ‘‘hydrophilic’’ drugs will be enhanced most by agents which have a positive impact on the SC diffusion process. Conversely, ‘‘lipophilic’’ drugs will be transported more efficiently if the enhancer can, in some way, facilitate the SC-viable tissue partitioning step.31 Some reports29,32 have shown that when using the lipophilic vehicle IPM, the SC did not seem to offer significant resistance although the viable ED is the rate-limiting barrier for the transport of relatively hydrophobic drugs. FP is lipophilic and practically insoluble in water (i.e., 0.14 mM33), indicating that IPM as a vehicle is fit to use in research. The effectiveness of different penetration enhancers on the permeation of FP through rat skin from IPM is shown in Figure 3. The corresponding parameters are given in Table 2. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
As seen from Table 2, the steady state flux and solubility of FP were all increased than control when penetration enhancers were used. AZ, MT, OA, SP, and NMP increased the Q8 h ( p < 0.05) with an ER of 2.26, 2.29, 2.49, 3.21, and 6.67, respectively. Among these penetration enhancers, NMP exerted the greatest solubilization on drug in the donor phase, in which the solubility of FP increased from 24.66 to 81.72 mg/mL and this is supported by previous studies.34 Akhter and Barry reported that NMP enhanced the skin permeation of FP and ibuprofen applied as solid drug film because the dissolution step was removed, and thermodynamic activity of the drug remaining in the solution increased after rapid permeation of NMP and NMP changed the diffusion resistance of the skin.35 In addition, the structure of NMP shows that there is a lactam group in the molecule, which is expected to exhibit H-bonding with FP.36 In our study, NMP showed the greatest solubilization among those penetration enhancers, which can be partially interpreted by H-bonding with the drug. However, OA, SP,
Figure 3. Effect of penetration enhancers on the permeation of FP through rat abdominal skin. (Each point represents the mean SE of three to five experiments.) DOI 10.1002/jps
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Table 2. Permeation Parameters of FP Through Rat Skin After Adding Different Penetration Enhancers Using an Identical Receiver Phase (PBS) and Donor Phases Consisting of IPM (n ¼ 3–5) Vehicle (w/w) IPM 5% AZ 5% MT 5% OA 5% SP 5% NMP
Q8 h (mg/cm2)
Flux (mg/cm2/h)
TLag (h)
Solubility (mg/mL)
p (cm/h 103)
ER
445.62 46.72 1006.52 71.61 1018.86 76.50 1108.14 225.64 1429.27 123.45 2973.37 244.45
69.01 7.45 145.15 7.55 162.54 8.62 153.60 28.12 248.47 20.83 474.21 46.64
1.53 0.11 1.15 0.11 1.80 0.19 0.89 0.34 2.29 0.25 1.61 0.31
24.66 44.86 31.33 28.79 29.47 81.72
2.80 3.24 5.19 5.34 8.43 5.80
— 2.26 2.29 2.49 3.21 6.67
and MT may preferentially formed intermolecular and intramolecular hydrogen bonds with autochthonous molecules, resulting in a decrease in the number of binding sites of hydrogen bond with drug.25
The Effect of Various Organic Amines on the Permeation of FP Since a lipophilic multicomponent system consisting of TEtA-10% EtOH/IPM system has been shown to selectively enhance the skin permeation of acidic drugs,23 and 10% EtOH/IPM system was suitable for the permeation of FP according to previous study. We investigated the effects of six organic amines (DEA, TEA, HEPP, EtA, DEtA, and TEtA) on the permeation of FP. The permeation profiles of FP in 10% EtOH/IPM binary solvent system obtained are shown in Figure 4.
Figure 4. Effect of various organic amines on the permeation of FP through rat abdominal skin. (Each point represents the mean SE of three to five experiments.) DOI 10.1002/jps
The corresponding permeation parameters are presented in Table 3. As illustrated in Figure 4 and Table 3, all the counter-ions employed in present study had an obviously positive effect on FP permeation ( p < 0.05). The Q8 h of FP increased to (9228.53 172.26) mg/cm2 in DEA, (7194.82 852.19) mg/cm2 in TEA, (6390.69 534.44) mg/ cm2 in EtA, (5566.21 194.95) mg/cm2 in TEtA, (4951.23 280.81) mg/cm2 in DEtA, (4562.55 730.59) mg/cm2 in HEPP, respectively. And the solubility of FP was increased markedly after adding organic amines, increasing the solubility of FP from 24.66 mg/mL in IPM to 118.81– 462.48 mg/mL in FP/amine-10% EtOH/IPM system. Bjerrum’s equation,37 which describes a critical separation distance for the formation of an ionpair, highlights the importance of the dielectric constant (e): a solvent with a high dielectric constant such as water (e ¼ 78.5) is unfavorable for ion-pair formation, while the interaction becomes increasingly important in solvents with e < 40.38 The donor vehicle consisted of 90% (w/w) IPM and 10% (w/w) EtOH, and the dielectric constant of the two solvents was 3.31 and 24.13, respectively;39 the calculated dielectric constant of the donor vehicle was 5.39.40–42 Thus, the 10% (w/w) EtOH/IPM employed in this research is suitable for evaluation of the effect of ionpairing on the percutaneous absorption of ionic drugs. 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. It is reported that the flux of drug decreased as the number of hydroxyls of the alkanolamine increased, indicating that the hydroxyl group has a negative effect on the permeation of drug. It has been reported that oxygen-containing monoterpenes may JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Table 3. Permeation Parameters of FP Through Rat Skin in the Presence of Various Counter-Ions Which Molar Quantities Were Equal to FP Dissolved in 10% EtOH/IPM(w/w) (n ¼ 3–5) Vehicle IPM 10% EtOH/IPM HEPP DEtA TEtA EtA TEA DEA
Q8 h (mg/cm2)
Flux (mg/cm2/h)
TLag (h)
Solubility (mg/mL)
p (cm/h 103)
ER
445.62 46.72 2453.38 157.47 4562.55 730.59 4951.23 280.81 5566.21 194.95 6390.69 534.44 7194.82 852.19 9228.53 172.26
69.01 7.45 372.6 45.12 751.54 169.82 496.16 12.28 752.45 10.73 759.19 120.86 1231.38 60.30 1297.53 121.81
1.53 0.11 1.04 0.17 1.67 0.54 0 0.76 0.20 0.46 0.46 2.11 0.60 0.89 0.70
24.66 58.18 152.01 118.81 248.28 306.50 462.48 257.81
2.80 6.40 4.94 4.18 3.03 2.47 2.66 5.03
— 5.51 10.24 11.11 12.49 14.34 16.15 20.71
preferentially form hydrogen with ceramide head groups of SC.43,44 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.45 Therefore, the hydroxyl group may inhibit the permeation of ion-pairs by forming hydrogen bonds with ceramide head groups. The physicochemical properties of various organic amines are summarized in Table 4. It was observed that some properties of organic amines could also affect their permeation-enhancing effect. The flux of FP was found to increase as a function of pKa value of the counter-ion except TEtA (Fig. 5), indicating 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, HEPP, TEtA are 11.1, 10.8, 9.5, 8.96, 9.04, and 7.76, respectively) and the acid (FP, pKa ¼ 4.2)46 leads to an almost complete shift of the protontransfer equilibrium of the O–H N $ O– H–
Nþ system.47 The larger the difference between the pKa of the amine and acid, the larger the attractive force between the amine and acid, and may lead to a high flux. TEtA has a low pKa but a relative high flux, this maybe because TEtA has the most hydroxyl groups among these organic amines, possibly there is a strong H-bonding action with FP just as the effect of NMP, which mainly lead to increasing the solubility and the permeation of FP. The Hansen solubility parameters of the counter-ions and FP were shown in Table 4 and calculated based on the methods of Hoftyzer/Van Krevelen and Hoy.27 Compounds with similar values for d are likely to be miscible. This is because the energy of mixing released by interactions within the components is balanced by the energy released by interaction between the components.48 Greenhalgh et al. classified excipients based on the difference between the solubility parameters of the excipients and drugs (Dd). The authors demonstrated that compounds with a Dd < 7.0 Mpa1/2 were likely to be miscible. When the Dd > 10.0 Mpa1/2 the compounds were likely to be immiscible. From the results
Table 4. Physicochemical Properties of the Organic Amines and FP
HEPP DEtA TEtA EtA TEA DEA FP
Formula
MW
Log Ko/w
pKa
da
C7H15NO NH(CH2 CH2OH)2 HOCH2CH2N(CH2 CH2OH)2 H2NCH2 CH2OH N(CH2CH3)3 NH(CH2CH3)2 C15H13FO2
129.20 105.14 149.19 61.08 101.19 73.14 244.26
0.96 1.43 1.00 1.31 1.45 0.58 3.86b
9.04 8.96 7.76 9.5 10.8 11.1 4.2c
24.24 28.87 30.42 27.28 16.10 15.83 22.05
Data were obtained from SRC PhysProp Database. a d Solubility parameter. b Morimoto et al.4 c Tang-Liu et al.46 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Figure 5. Relationship between the flux of FP in the presence of counter-ions except TEtA from 10% EtOH/ IPM (w/w) and the pKa of the counter-ions. (Each point represents the mean of three to five experiments.)
summarized in Table 4 the amines (d for DEA, TEA, EtA, TEtA, DEtA, HEPP, are 15.83, 16.10, 27.28, 30.42, 28.87, 24.24, respectively) and FP (d ¼ 22.05) in this study should show some miscibility with a range of Dd from 2.19 to 8.37. An inverse relationship between the flux and d of the counter-ions is shown in Figure 6. Then based on the good miscibility between the drug and the counter-ions, the counter-ion with a decrease in solubility parameter (an increase in lipophilicity) exhibited an increase in the flux of FP. Such behavior could be elucidated by ion-pair formation and the partition between the SC and the ED.29 The excised rat skin contains the ED (divided into the SC and the viable ED) and the dermis. The viable ED and dermis (ED) are similar and regarded as an aqueous protein gel.49 Drugs penetrate the skin according to the following
Figure 6. Relationship between the flux of FP in the presence of six counter-ions from 10% EtOH/IPM (w/w) and the d (solubility parameter) of the counter-ions. (Each point represents the mean of three to five experiments.) DOI 10.1002/jps
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process, during which drugs partition between the EI system and the SC firstly, and then diffuse through the SC. Subsequently, they partition between the SC and the ED, and finally diffuse through the ED. Once they reach the dermis, drugs are absorbed by the blood capillaries within minutes. Lipophilic drugs pass through the SC, which is essentially a lipid layer, and then they must transfer directly into the aqueous medium— ED, otherwise they will remain in the SC. According to the theory, the main rate-limiting step for lipophilic drug is the partition from the SC to the ED. So the affinity for the ED, in other words the hydrophilicity, is an important factor to lipophilic drug. This has also been confirmed by the study of Jiang et al.50 Therefore, improving the hydrophilicity of lipophilic drug may facilitate the partition between the SC to the ED. Addition of counter-ions is reported to be an effective method to obtain a balanced hydrophilicity– lipophilicity for both hydrophilic and lipophilic drugs. In case of a hydrophilic drug, such as 5aminolevulinic acid51 and methotrexate,52 a lipophilic counter-ion can be used to enhance the lipophilicity of the drug and hence promote the SC diffusion process. In contrast a hydrophilic counter-ion should be used for lipophilic drugs, such as benzydamine53 and mefenamic acid.29 Therefore, ‘‘lipophilic’’ drugs will be transported more efficiently if the counter-ion can, in some way, facilitate the SC-viable tissue partitioning step.31 It agreed with the present study that addition of hydrophilic counter-ions improved the hydrophilicity of FP, and then facilitates the SC– ED partitioning step. After adding the hydrophilic counter-ions, the ‘‘ion-pair’’ as a unit showed the similar characteristic as hydrophilic drugs, since the ‘‘ion-pair’’ had an increase in affinity (lipophilicity) to SC exhibited an increase in the flux of FP. As indicated above, the enhancing effect of the counter-ions is greater than that of penetration enhancers. Since FP is a lipophilic drug, the viable ED, not the SC, is the rate-limiting step in skin penetration.29,54,55 Penetration enhancers may mainly increase the transdermal drug penetration by altering the barrier function of the SC. Counter-ions may decrease the lipophilicity of FP by forming an ion-pair and hence may promote the key step of the partition of FP into the ED. The penetration of FP is determined by the viable ED, so the counter-ions are more effective than penetration enhancers for increasing the permeation of FP through rat skin. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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Table 5.
a b c d e f g h i j
1
H NMR Chemical Shifts of FP and its Ion-Pairs for Proton on Carbon
FP
DEA
TEA
EtA
TEtA
DEtA
HEPP
7.22 7.24 7.41 7.47 7.49 7.54 3.78 1.41 Overlap Overlap
7.19 7.20 7.37 7.45 7.47 7.52 3.52 (þ0.26) 1.33 (þ0.08) Overlap Overlap
7.21 7.22 7.40 7.45 7.47 7.53 3.66 (þ0.12) 1.37 (þ0.04) Overlap Overlap
7.17 7.19 7.37 7.45 7.47 7.52 3.40 (þ0.38) 1.31 (þ0.10) Overlap Overlap
7.22 7.24 7.41 7.47 7.49 7.54 3.75 (þ0.03) 1.40 (þ0.01) Overlap Overlap
7.19 7.20 7.39 7.45 7.47 7.52 3.52 (þ0.26) 1.34 (þ0.07) Overlap Overlap
7.21 7.23 7.40 7.46 7.48 7.53 3.68 (þ0.10) 1.38 (þ0.03) Overlap Overlap
NMR Discussion The goal of the 1H-NMR measurement was to obtain evidence for the presence of ion-pair formation between FP and respective counterions from the chemical shift changes to protons near the carboxyl group, as ion-pair formation is an indicator of increased permeability of the counter-ions through skin. Table 5 shows the modulation of the FP assigned protons signals in the different formulations. The changes of chemical shifts of the ionpairs relative to FP are obviously indicated in Figure 7. It is clear that addition of equimolar counter-ions to FP resulted in no significant shifts of aromatic protons having shifts of only 0.01– 0.05 ppm. However, the (g) and (h) protons have obviously changed in chemical shifts, which have been the focus of this study. Assignment (g) and (h) represent a single proton and a methyl group (three equivalent protons), respectively, which attached to the carboxyl group. The proton (h) shifted upfield by 0.01–0.08 ppm, while the proton (g) signal was considerably shifted upfield by 0.03–0.38 ppm. The high shifts exhibited by protons g and h suggest that these groups were
particularly influenced by bonding between counter-ions and FP, which is indicative of ion-pair formation. It could be elucidated that to FP molecule, the carboxyl group plays a role of electrophilic action on proton (g) and (h), which leads to a decrease of electron atmosphere density and a deshielding. After application of counterions, the carboxyl group of FP salified with respective bases, which decreased the electrophilic action, thus, the proton (g) and (h) shifted upfield. Similar bonding involving carboxylic acids and amine groups has been reported by Hosono et al.56; Sanders and Hunter57; Beten et al.58; Servet;59 and Sarveiya et al.24
CONCLUSION This work evaluated the enhancing effect of counter-ions on FP as well as penetration enhancers and proved the formation of ion-pairs. We can conclude that counter-ions are more efficient than penetration enhancers with a combination of 10% EtOH/IPM system. Of all the amines studied, DEA produced the greatest
Figure 7. The chemical shifts of the assigned protons of FP. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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enhancement of FP, which may be influenced by pKa, its structure and hydrophilicity. Among the penetration enhancers investigated, NMP was found to be the most promising chemical enhancer, attributed to the altering barrier function of SC and its solubilization on FP. We will synthesis various FP–amine complexes, and optimize the formulation of these complexes in the form of patch in further research.
10.
11.
12.
ACKNOWLEDGMENTS The authors are grateful to Professor Yasunori Morimoto (Faculty of Pharmaceutical Sciences, Josai University, Japan) for his kind gift of twochamber diffusion cells and a synchronous motor.
REFERENCES 1. Lee DJ, Burt CT, Koch RL. 1992. Percutaneous absorption of flurbiprofen in the hairless rat measured in vivo using 19F magnetic resonance spectroscopy. J Invest Dermatol 99:431–434. 2. Poul J, West J, Buchanan N, Grahame R. 1993. Local action transcutaneous flurbiprofen in the treatment of soft tissue rheumatism. Br J Pharmatol 32:1000–1003. 3. Heyneman CA, Lawless-Liday C, Wall GC. 2000. Oral versus topical NSAID in rheumatic disease: A comparison. Drugs 60:555–574. 4. Morimoto Y, Hatanaka T, Sugibayashi K, Omiya H. 1992. Prediction of skin permeability of drugs: Comparison of human and hairless rat skin. J Pharm Pharmacol 44:634–639. 5. Bronaugh RL, Stewart RF. 1984. Method for in vitro percutaneous absorption studies III: Hydrophobic compounds. J Pharm Sci 73:1255–1258. 6. Bronaugh RL, Stewart RF. 1986. Method for in vitro percutaneous absorption studies VI: Preparation of the barrier layer. J Pharm Sci 75:487– 491. 7. Fang JY, Hwang TL, Fang CL, Chiu HC. 2003. In vitro and in vivo evaluations of the efficacy and safety of skin permeation enhancers using flurbiprofen as a model drug. Int J Pharm 255: 153–166. 8. Fang JY, Chiu HC, Wu JT, Chiang YR, Hsuc SH., 2004. Fatty acids in Botryococcus braunii accelerate topical delivery of flurbiprofen into and across skin. Int J Pharm 276:163–173. 9. Charoo NA, Shamsher AAA, Kohli K, Pillai K, Rahman Z. 2008. Improvement in bioavailability of
DOI 10.1002/jps
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
1835
transdermally applied flurbiprofen using tulsi (Ocimum sanctum) and turpentine oil. Colloids Surf B Biointerfaces 65:300–307. Swart H, Breytenbach JC, Hadgraft J, Plessis J. 2005. Synthesis and transdermal penetration of NSAID glycoside esters. Int J Pharm 301:71– 79. Jain SK, Chourasia MK, Masuriha R, Soni V, Jain A, Jain NK, Gupta Y. 2005. Solid lipid nanoparticles bearing flurbiprofen for transdermal delivery. Drug Deliv 12:207–215. Valenta C, Siman U, Kratzel M, Hadgraft J. 2000. The dermal delivery of lignocaine: Influence of ion pairing. Int J Pharm 20:77–85. Hatanaka T, Kamon T, Morigaki S, Katayama K, Koizumi T. 2000. Ion pair skin transport of a zwitterionic drug, cephalexin. J Control Release 66:63– 71. Trotta M, Ugazio E, Peira E, Pulitano C. 2003. Influence of ion pairing on topical delivery of retinoic acid from microemulsions. J Control Release 86:315–321. Wilson TH, Wiseman G. 1954. The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J Physiol 123:116–125. Gasco M, Eandi M. 1984. The influence of bile salts on the absorption in vitro and in vivo of propranolol. Pharm Biomed Anal 2:424–428. Hadgraft J, Walters KA, Wotton PK. 1985. Facilitated transport of sodium salicylate across an artificial lipid membrane by Azone. J Pharm Pharmacol 37:725–727. Hadgraft J, Walters KA, Wotton PK. 1986. Facilitated percutaneous absorption: A comparison and evaluation of two in vitro models. Int J Pharm 32:257–263. Young C-S, Shi C-K, Lee M-H, Kim S-K. 1988. Effect of sodium salicylate on in vitro percutaneous penetration of isopropamide iodide through mouse skin. Int J Pharm 45:59–64. Pedersen M. 1990. Synergistic action of clotrimazole and certain anionic surfactants may be due to ion pair formation. Acta Pharm Nord 2:367– 370. Megwa SA, Cross SE, Benson HAE, Roberts MS. 2000. Ion-pair formation as a strategy to enhance topical delivery of salicylic acid. J Pharm Pharmacol 52:919–928. Megwa SA, Cross SE, Whitehouse MW, Benson HAE, Roberts MS. 2000. Effect of ion pairing with alkylamines on the in-vitro dermal penetration and local tissue disposition of salicylates. J Pharm Pharmacol 52:929–940. Fang L, Kobayashi Y, Numajiri S, Kobayashi D, Sugibayashi K, Morimoto Y. 2002. The enhancing effect of a triethanolamine-ethanol-isopropyl myr-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
1836
24.
25.
26.
27. 28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
MA ET AL.
istate mixed system on the skin permeation of acidic drugs. Biol Pharm Bull 25:1339–1344. Sarveiya V, Templeton JF, Benson HAE. 2004. Ionpairs of ibuprofen: Increased membrane diffusion. J Pharm Pharmacol 56:717–724. Wang ML, Fang L, Ren CS, Li T. 2008. Effect of ionpairing and enhancers on scutellarin skin permeability. J Pharm Pharmacol 60:429–435. Maitre MM, Longhi MR, Gladys GG. 2007. Ternary complexes of flurbiprofen with HP-b-CD and ethanolamines characterization and transdermal delivery. Drug Dev Ind Pharm 33:311–326. Krevelen V, Krevelen DW. 1990. Properties of Polymers. Amsterdam: Elsevier Science Publishing Co. Hancock BC, York P, Rowe RC. 1997. The use of solubility parameters in pharmaceutical solid dosage form design. Int J Pharm 148:1–21. Fang L, Numajiri S, Kobayashi D, Morimoto Y. 2003. The use of complexation with alkanolamines to facilitate skin permeation of mefenamic acid. Int J Pharm 262:13–22. McDaid DM, Deasy PB. 1996. Formulation development of a transdermal drug delivery system for amlodipine base. Int J Pharm 133:71–83. Hori M, Satoh S, Maibach HI, Guy RH. 1991. Enhancement of propranolol hydrochloride and diazepam skin absorption in vitro: Effect of enhancer lipophilicity. J Pharm Sci 80:32–35. Wenkers BP, Lippold BC. 1999. Skin penetration of non-steroidal anti-inflammatory drugs out of a lipophilic vehicle: Influence of the viable epidermis. J Pharm Sci 88:1326–1331. Tavornvipas S, Hirayama F, Arima H, Uekama K, Ishiguro T, Oka M, Hamayasu K, Hashimoto H. 2002. 6-O-a-(4-O-a-D-glucuronyl) -D-glucosyl-ßcyclodextrin: Solubilizing ability and some cellular effects. Int J Pharm 249:199–209. Sawa CL, Heng PW, Chin WW, Soo KC, Olivo M. 2006. Enhanced photodynamic activity of hypericin by penetration enhancer N-methyl pyrrolidone formulations in the chick chorioallantoic membrane model. Cancer Lett 238:104–110. Akhter S, Barry BW. 1985. Absorption through human skin of ibuprofen and flurbiprofen; effect of dose variation, deposited drug film, occlusion and the penetration enhancer N-methyl-2-pyrrolidone. J Pharm Pharmacol 37:27–37. Lee PJ, Ahmad N, Langer R, Mitragotri S, Prasad SV. 2006. Evaluation of chemical enhancers in the transdermal delivery of lidocaine. Int J Pharm 308: 33–39. Hatanaka T, Kamon T, Morigaki S, Katayama K, Koizumi T. 2000. Ion pair skin transport of a zwitterionic drug, cephalexin. J Control Release 66:63– 71. Kraus CA. 1956. The ion pair concept: Its evolution and some applications. J Phys Chem 60:129– 141.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
39. Harada S, Takahashi Y, Nakagawa H, Yanmashita F, Hashida MB. 2000. Effect of vehicle properties on skin penetration of emedastine. Pharm Bull 23: 1224–1228. 40. Prakongpan S, Nagai T. 1984. Solubility of acetaminophen in cosolvents. Chem Pharm Bull 32:340– 343. 41. Chien YW. 1984. Solubilization of metronidazole by watermiscible multi-cosolvents and water-soluble vitamins. J Parent Sci Technol 38:32–36. 42. Dumanovic D, Kosanovic DJ, Ardakovic D, Jovanovic J. 1992. Solubilization of ipronidazole by co-solvents. Pharmazie 47:603–607. 43. Jain AK, Narishetty STK, Panchagnula R. 2002. Transdermal drug delivery of imipramine hydrochloride. I. Effect of terpenes. J Control Release 79:93–101. 44. Narishetty STK, Panchagnula R. 2004. Transdermal delivery of zidovudine: Effect of terpenes and their mechanism of action. J Control Release 95: 367–379. 45. Potts RO, Guy RH. 1995. A predictive algorithm for skin permeability: The effects of molecular size and hydrogen bond activity. Pharm Res 12:1628–1633. 46. Tang-Liu DD, Liu S. 1987. Relationship between the ocular and systemic disposition of flurbiprofen: The effect of altered protein dynamics at steady state. J Pharm and Bio 15:387–397. 47. Ratajczak H, Sobczyk L. 1969. Dipole moments of hydrogen-bonded complexes and proton-transfer effect. J Chem Phys 50:556–557. 48. Greenhalgh D, Williams A, Timmins P, York P. 1999. Solubility parameters as predictors of miscibility in solid disperisons. J Pharm Sci 88:1182–1190. 49. Scheuplein RJ. 1976. Mechanism of percutaneous absorption II. Transient diffusion and the relative importance of various routes of skin permeation. J Invest Dermatol 48:79–88. 50. Jiang Y, Fang L, Niu X, Ma R, He Z. 2008. The effect of ion pairing on the skin permeation of amlodipine. Pharmazie 63:356–360. 51. Auner BG, Valenta C, Hadgraft J. 2003. Influence of lipophilic counter-ions in combination with phloretin and 6-ketocholestanol on the skin permeation of 5-aminolevulinic acid. Int J Pharm 255:109–116. 52. Trotta M, Pattarino F, Gasco MR. 1996. Influence of counter ions on the skin permeation of methotrexate from water-oil microemulsions. Pharm Acta Helv 71:135–140. 53. Sarveiya V, Templeton JF, Benson HAE. 2005. Effect of lipophilic counterions on membrane diffusion of benzydamine. Eur J Pharm Sci 26:39–46. 54. Wenkers BP, Lippold BC. 1999. Skin penetration of non-steroidal antiinflammatory drugs out of a lipophilic vehicle: Influence of the viable epidermis. J Pharm Sci 88:1326–1331. 55. Harada S, Takahashi Y, Nakagawa H, Yanmashita F, Hashida M. 2000. Effect of vehicle properties on
DOI 10.1002/jps
EFFECT OF COUNTER-IONS AND PENETRATION ENHANCERS
skin penetration of emedastine. Biol Pharm Bull 23:1224–1228. 56. Hosono T, Tsuchiya S, Matsumaru H. 1979. Molecular complex formation between N-vinyl-2-pyrrolidone and ajmaline. Chem Pharm Bull 27:58–64. 57. Sanders JKM, Hunter BK. 1989. Modern NMR Spectroscopy. Oxford: Oxford University Press. p. 215.
DOI 10.1002/jps
1837
58. Beten DB, Gelbcke M, Diallo B, Moes AJ. 1992. Interaction between dipyridamole and Eudragit, S. Int J Pharm 88:31–37. 59. Servet B, Nadir B, Rytting JH. 1996. Interaction of indomethacin with a new penetration enhancer, dodecyl 2-(N,N-dimethylamino)propionate (DDAIP): Its effect on transdermal delivery. Int J Pharm 127:245–253.
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