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Transdermal drug delivery of imipramine hydrochloride.
Journal of Controlled Release 79 (2002) 93–101 www.elsevier.com / locate / jconrel
Transdermal drug delivery of imipramine hydrochloride. I. Effect of terpenes Amit Kumar Jain, Narisetty Sunil Thomas, Ramesh Panchagnula* Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research ( NIPER), Sector 67, Phase-X, Mohali-160062, Punjab, India Received 14 February 2001; accepted 17 October 2001
Abstract The objective of this investigation was to study the effect of different terpenes on IMH permeation in EtOH:W (2:1) system. Permeation studies of IMH were carried out with unjacketed Franz diffusion cells through rat skin. The flux of IMH with terpenes was found to be significantly higher than that in control (EtOH:W, 2:1) (P,0.05). Amongst all studied terpenes, menthol, terpineol, cineole and menthone were found to be effective permeation enhancers for IMH. It was found that the contribution of diffusivity in enhanced permeation of IMH was much higher in comparison to partitioning of IMH in skin with terpene treatment. Results of this study were explained with the help of H-bond breaking potential and self-association of terpenes. In order to elucidate the effect of terpenes on stratum corneum barrier FT-IR was used. 2002 Elsevier Science B.V. All rights reserved. Keywords: Permeation enhancer; Terpene; FT-IR; Imipramine hydrochloride
1. Introduction TCAs are widely used in treatment of unipolar and bipolar depression, which is characterized by extreme sadness, despair and anhedonia. The problem of high first-pass metabolism, variation in gastroinAbbreviations: DCR, diffusion coefficient ratio; ER, enhancement ratio; EtOH, ethanol; FT-IR, Fourier-transform infrared spectroscopy; IMH, imipramine hydrochloride; PCR, partition coefficient ratio; SC, stratum corneum; SNK, Student–Newman– Keuls; TCAs, tricyclic antidepressants; TDDS, transdermal drug delivery system; W, water *Corresponding author. Tel.: 191-172-214-682 / 688; fax: 191-172-214-692. E-mail address: [email protected] (R. Panchagnula).
testinal absorption [1] and patient non-compliance with conventional per oral delivery can be overcome with transdermal delivery of TCAs. As transdermal delivery offer inherent advantages such as: (a) bypasses first pass metabolism; (b) enables control of input; (c) avoids problems of stomach emptying, pH effects and enzymatic deactivation associated with gastrointestinal tract passage [2]. However, their poor permeability and high dose are the major hurdles in delivery across the skin. Panchagnula [3] and Stott et al. [4] have suggested the use of solvent, e.g., dimethylsulfoxide, dimethylacetamide and complex coacervation technique, respectively, for enhancement of TCAs permeation through skin [3,4]. With these approaches flux of IMH was not high
0168-3659 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00524-7
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enough to achieve the desired plasma level. Therefore, in this investigation terpenes were selected as permeation enhancers to study their effect on IMH in vitro permeation across the rat skin. Terpenes are non-toxic and non-irritant [5] to skin and are extensively used in transdermal delivery as permeation enhancers with hydrophilic (5-fluorouracil [6], propanolol hydrochloride [7]) and lipophilic (indomethacin and ketoprofen [8], estradiol [9]) drugs. In addition, FT-IR was used to elucidate the mechanism of action of terpenes effect on lipid bilayer of SC.
2. Materials IMH and cineole were procured from Sigma (St. Louis, MO, USA). [ 3 H]IMH (specific activity, 48.00 Ci / mmol) was obtained from Du Pont (Wilmington, DE). Pulegone, a-terpineol, menthol, ethanol, carvone and menthone were purchased either from Merck, (Germany) or Fluka Chem (Germany). Tissue solubilizer (NCS II) and scintillation cocktail (BCS 104) were purchased from Amersham (UK). All other chemicals used were of reagent grade.
3. Methods
3.1. Preparation of full thickness skin The skin samples were harvested from the dorsal surface of the Sprague–Dawley rats. Hair on dorsal skin of animal was removed with animal hair clipper (Aesculp, Germany), subcutaneous tissue was surgically removed and dermis side was wiped with isopropyl alcohol to remove residual adhering fat. The skin was washed with distilled water, wrapped in aluminium foil and stored in a freezer at 2208C till further use. The protocol for this study was approved by NIPER’s Institutional Animal Ethics Committee.
3.2. Separation of epidermis from full thickness skin Frozen skin was thawed at room temperature and treated with 2 M sodium bromide solution in water
for 6 h [10]. The epidermis was separated by using a cotton swab moistened with distilled water. Epidermal sheet was cleaned by washing with distilled water and dried under vacuum. Dried epidermal sheets were stored in desiccator until further use.
3.3. Ex vivo permeation study IMH permeation across the skin was determined (n54) using unjacketed Franz cells with a diffusional surface area of 0.785 cm 2 . The cells were placed on dry heater and stirrer block supplied by Permegear (USA). Phosphate buffer (pH 7.4), containing 0.01% (w / v) sodium azide as a preservative was used as the receptor medium. The receptor fluid was sonicated to remove dissolved gases and equilibrated at 378C before placing in the receptor compartment. Cells were filled with receptor fluid and skin was mounted on diffusion cell with dermal side in contact with the receptor medium, then equilibrated at 378C for 8–10 h. IMH solution (200 ml of 75 mg / ml and spiked with 1 mCi / ml) in solvent system, i.e., EtOH:W (2:1) with or without terpene (5%, w / v) was applied to SC side in the donor compartment. Donor compartment was covered with paraffin film. After application of drug solution, samples (200 ml) were withdrawn from the receptor compartment at different time intervals and analysed for drug content by radiochemical analysis. Sample volume was immediately replaced with fresh receptor medium after each sampling.
3.4. Determination of partition coefficient Partition coefficient of IMH at 32618C between EtOH:W (2:1) system with or without terpene and epidermal sheet were determined in triplicate. Epidermal sheets were cut into 1-cm 2 pieces and each piece was dipped into separate screw cap vials containing 1 ml of water with 0.01% (w / v) sodium azide, for 36 h. Epidermal sheets were then blotted dry, weighed and soaked into 1 ml of various solvent systems containing IMH (1.16 mg / ml with 0.016 mCi / ml). All vials were kept under constant shaking in water bath (Julabo, Germany) at 328C for 12 h. Epidermal sheets were then blotted dry, transferred to glass scintillation vials and solubilized in 600 ml of tissue solubilizer. Five ml of scintillation cocktail
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and 100 ml of glacial acetic acid were added into each glass scintillation vial [6]. After 12 h, vials were counted for radioactivity in liquid scintillation counter (Wallac 1405, Finland). Radioactivity left in the solvents in which epidermal sheets were soaked was also counted in duplicate for mass balance.
3.5. FT-IR Dried epidermal sheet was cut into approximately 2-cm 2 pieces and soaked in water containing 0.01% of sodium azide for 36 h. Epidermal pieces were dried under ambient condition for overnight and then kept under vacuum in phosphorus pentoxide (P2 O 5 ) desiccators for 15 min to ensure complete removal of free water. FT-IR spectra of each epidermal piece were recorded before terpene treatment (control) and immediately after this, pieces were dipped into respective terpene solutions (5%, w / v) in EtOH:W (2:1) system for 12 h. Each piece after treatment was washed three times with EtOH:W (1:1) and water, then air dried for 2 h. Samples were kept under vacuum in phosphorus pentoxide (P2 O 5 ) desiccators for 15 min to remove alcohol and free terpenes completely. FT-IR spectra of all pieces treated with terpenes were recorded on Nicolet (USA) FT-IR spectrophotometer with following acquisition parameters; resolution of 2 cm 21 , number of scans was 100 and detector was DTGS. All epidermal pieces after drying were kept under vacuum for 36 h and FT-IR spectra were recorded again to ensure that terpenes remained on surface after treatment were removed completely by washing and were not interfering with skin lipid and protein peaks.
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SSF Permeability 5 ]] Cd where, SSF is steady-state flux (mg cm 22 h 21 ), Cd is concentration in donor compartment. Since there is a possibility of unpredictable alteration in diffusion pathlength (h) after terpene treatment, therefore, in order for better approximation of diffusivity (D) of IMH, D/h 2 was derived from lag time by following equation [11]; D/h 2 5 1 /(6 ? t lag ) where, t lag is lag time in hours.
4. Results and discussion Permeation profile of IMH with and without the treatment of terpenes is shown in Fig. 1. Flux values of IMH, with and without terpene treatment are given in Table 1, in decreasing order is as follows; menthol$cineole.terpineol.menthone. pulegone.carvone.control (EtOH:W, 2:1). Flux of IMH in terpenes was significantly different from control (EtOH:W, 2:1) (P,0.05). However, among the terpenes, flux of IMH with menthol, cineole and
3.6. Data analysis Cartesian plots with cumulative amount of drug present in receptor compartment versus time were plotted. Flux values (mg cm 22 h 21 ) were calculated from the slopes of the steady-state portion of the plots. Lag times were calculated from the intercepts of extrapolated steady-state flux to the x-axis. Permeability values were calculated by the following equation;
Fig. 1. Effect of different class of terpenes on IMH permeation profile.
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Table 1 Permeation parameters of IMH with and without terpene treatment, mean (6S.D.) Chemical class Control Alcohol Ether Ketone
E:W (2:1) Menthol Terpeneol Cineole Menthone Pulegone Carvone
J (mg cm 22 h 21 )
Lag time (h)
Kp 3(10 3 ) (cm/h)
P
45.7 (18.80) 754.0* (159.60) 530.0* (188.00) 623.0* (97.80) 452.2* (90.50) 231.5* (40.80) 288.7* (30.50)
32.29 (13.52) 5.81* (0.82) 6.54* (2.38) 5.43* (2.36) 4.69* (1.19) 9.78* (1.39) 11.46* (1.87)
0.61 10.50 7.06 8.03 6.02 3.08 3.84
2.04 1.90 2.70 3.19 1.91 2.73 1.84
(0.62) (0.29) (0.44) (0.45) (0.13) (1.53) (0.20)’
D/h 2 (h 21 )
PCR
DCR
ER
0.0059 (0.002) 0.0290 (0.003) 0.0285 (0.011) 0.0392 (0.026) 0.0378 (0.012) 0.0151 (0.002) 0.0172 (0.002)
– 0.93 (0.14) 1.32 (0.21) 1.56 (0.22) 0.93 (0.06) 1.34 (0.75) 0.90 (0.09)
– 4.92 (0.63) 4.83 (1.93) 6.65 (4.54) 6.42 (2.14) 2.56 (0.44) 2.93 (0.42)
– 16.69 11.58 13.61 9.80 5.03 6.27
*Significantly different from control, P,0.05. PCR, partition coefficient with terpene treatment / partition coefficient without terpene treatment; DCR, diffusivity with terpene treatment / diffusivity without terpene treatment; and ER, flux with terpene treatment / flux without terpene treatment.
terpineol was significantly different from pulegone and carvone (P,0.05), whereas flux of IMH in menthol and cineole were comparable. Lag time of IMH with terpene treatment was significantly reduced as compared to control (EtOH:W, 2:1) (P,0.05) and order of terpene effect on lag time in decreasing order is as follows control.carvone.pulegone.terpineol.menthol. cineole.menthone. The main barrier or rate-limiting step in transdermal drug delivery of polar, water-soluble drug such as IMH is the lipophilic part of SC, in which lipids (ceramides) are arranged in a bilayer. Ceramides (especially ceramide 2 and ceramide 5, which are abundantly present in SC) are tightly packed in the bilayer due to the high degree of hydrogen bonding. The amide I group of ceramide is hydrogen bonded to amide I group of another ceramide and a tight network of hydrogen bonding is formed at the head of ceramides. This hydrogen bonding provides strength and stability to lipid bilayer and imparts the barrier property to SC. Recently, it has been proved with model SC lipids (hexadecanoic acid, cholesterol, bovine non-hydroxy fatty acid ceramide and bovine hydroxy fatty acid ceramide) that amide I of ceramide 5 is laterally hydrogen bonded between head of ceramides [12]. In addition, amide I of ceramide 2 is responsible for a high degree of lateral and transverse hydrogen bonding, which involves amide I group and carbonyl moiety of opposite ceramide and fatty acid, respectively. When skin is treated with terpenes, the existing network of hydrogen bonds between ceramides may get loosened
because of ‘competitive hydrogen bonding’ (Fig. 2). The hydrogen bond network at the head of ceramides breaks as terpenes (e.g., menthol, terpineol) enter into the lipid bilayer of SC, since alcoholic –OH group is more electronegative than –NH of amide I [13]. Since alcoholic –OH group can accept or donate the hydrogen bond, it leads to disruption of existing hydrogen bonding between ceramides head groups, thereby facilitating the permeation of IMH and is also reflected by high ER with menthol and terpineol (Table 1). However, in case of menthone, pulegone, carvone and cineole, only hydrogen bond accepting moieties, viz. carbonyl, and ether groups are present, respectively. The extent of disruption of the hydrogen bond network between the ceramides heads is less, as is evident from relatively less ER with pulegone and carvone (Table 1). With cineole and menthone, ER and DCR were found to be higher than that of pulegone and carvone. Of all terpenes studied, the boiling point (bp) of cineole (bp 1738C) and menthone (2108C) is approximately 408C and 2–178C lesser than that of other terpenes, respectively, including menthol (bp 2158C) and terpineol (bp 2178C). This is an indication of weak cohesive or self-association of cineole and menthone molecules [14] or in other words oxygen of functional ether and carbonyl group is in free form for interaction. Therefore, the energy required for competitive hydrogen bonding in skin ceramides is relatively less for cineole and menthone, which can be attributed to the higher ER and DCR as found with these terpenes. On the other hand, in case of menthol,
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Fig. 2. Mechanistic aspects of terpenes action on lipid bilayer of SC.
terpineol, pulegone and carvone, additional energy is required to free the respective functional group from strong self-association, as reflected by higher boiling point (Table 2). Hence low DCR was found with menthol and terpineol. Whereas in case of pulegone and carvone self-association and presence of only hydrogen bond acceptor is responsible for weak interaction with skin ceramides, lower ER and DCR were found as compared to other terpenes. Menthone, pulegone and carvone belong to same class of terpene, i.e., ketone terpene (carbonyl group), but menthone was found to be an effective permeation enhancer for IMH when compared to the other two ketone terpenes. This difference in effectiveness of ketone terpenes as permeation enhancers may be because of different degrees of unsaturation in Table 2 Boiling point of terpenes Terpene
Boiling point (8C)a
Menthol Cineole Terpineol Menthone Pulegone Carvone
215 173 217 210 224 230
a
Boiling points of terpenes are taken from Merck and Aldrich catalogues.
ketone terpenes, e.g., menthone (0), pulegone (2) and carvone (3); (degree of unsaturation), resulting in difference in cohesiveness as is also indicated by their boiling point (Table 2). Partition coefficients of IMH in epidermis / vehicle (EtOH:W (2:1) with or without terpene) system were determined and results are shown in Table 1. Results of this study indicate that the contribution of partitioning in enhanced permeation of IMH after terpene treatment is negligible, as shown by lower PCR values, and are in agreement with previous finding [6,15]. FT-IR of rat epidermis after 12 h treatment shows no shift in wavenumber (Fig. 3a) and changes produced in peak height (2850 and 2920 cm 21 ) and area (2850 and 2920 cm 21 ) does not significantly differ from changes produced by the EtOH:W (2:1) system (results not shown). The EtOH:W (2:1) system with or without terpenes was found to be inefficient to extract the lipid from rat epidermis, as is evident from presence of –C5O peak of ester at 1740 cm 21 region (Fig. 3b). As discussed earlier ceramide 2 and 5 form H-bonds between each other in the SC lipid bilayer and ceramide 2 is responsible for transverse H-bonding in the lipid bilayer, thus causing the split in peak at 1650 cm 21 (amide I) which was found in model membrane prepared from SC lipids [12]. A similar split in the peak at 1650 cm 21 region was observed in our control experi-
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Fig. 3. FT-IR spectra of rat epidermis. (A) After 12 h treatment with (a) water, (b) EtOH:W (2:1), (c) cineole, (d) menthol, (e) terpineol, (f) menthone, (g) pulegone, (h) carvone. (B) Individual control before treatment with different terpenes (a) EtOH:W (2:1), (b) cineole, (c) menthol, (d) terpineol, (e) menthone, (f) pulegone, (g) carvone. (C) After 12 h treatment with different class of terpenes.
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Fig. 3. (continued)
ments also (Fig. 3b). After treatment with terpenes, only a single peak at 1650 cm 21 was found (Fig. 3c), which indicates breaking of H-bonds by terpenes. However, the possibility of breaking of H-bond network existing in a-keratin and thereby expansion of corneocytes cannot be overlooked for the EtOH:W (2:1) system because the split at 1650 cm 21 also disappeared in this system. Since the main barrier in permeation is the lipid bilayer, this effect may not be significant, unless the lipid bilayer fluidity increases [16] as is indicated by lower flux values with the EtOH:W (2:1) system. With ethanol, bilayer fluidity is not increased as found by DSC studies from literature and there was no shift in lipid transition temperature after ethanol treatment [17]. However, with terpenes in the EtOH:W (2:1) system, apart from the EtOH:W effect on a-keratin structure, increased lipid bilayer fluidity by terpenes may also be responsible for higher ER. Since it was observed that the FT-IR could not differentiate the effect of EtOH:W (2:1) and terpenes on protein and lipid bilayer of epidermis, respectively, therefore DSC and
ESR data from literature was used to support the assumption of breaking of H-bonds between the head group of ceramides on treatment with terpenes. With DSC, It has been reported that cineole and menthone induce shifts in transition temperature along with increased entropy (measure of disorder) at physiological temperature or below transition temperature [15]. This is attributed to loosening of highly organised lipid bilayer structure due to breaking of the H-bond network. The hypothesis of breaking or loosening of the H-bond network between ceramides head groups by –OH or –C=O group of terpenes can also be supplemented by the effect of limonene (terpene without –OH or –C=O group) on the entropy of lipid transition. Though it has been reported that limonene caused a shift in transition temperature of skin, entropy associated with lipid transition was found to be unchanged [15,18]. In other words, limonene is not able to break the Hbond network between ceramides due to absence of a hydrogen bond accepting or donating group. It has been reported that ER for 5-fluorouracil (a polar,
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hydrophilic) with limonene was less than that of cineole, menthone and nerolidol. With ESR, it has been reported that the fluidity of the lipid bilayer is increased after menthol treatment [19]. It has been reported that cineole forms a pool in model SC matrix and enhances the permeation of 5-fluorouracil and oestradiol [20]. At 5% (w / v) concentration, no characteristic peak of terpene was found in FT-IR spectra of rat epidermis after 12 h treatment with terpene, which implies that at this concentration terpenes may not form a pool inside the lipid bilayer of SC. As described in this study, permeation of a watersoluble, polar drug such as IMH can be increased significantly through breaking of hydrogen bonds between ceramide heads by terpenes with weak selfassociation or which have the ability of donating or accepting H-bond.
5. Conclusions Amongst all the terpenes, menthol was found to be an effective permeation enhancer for IMH. In general, it was observed that terpenes with minimum degree of unsaturation, like menthol, cineole, menthone, are good permeation enhancers for polar and water-soluble drugs, such as IMH. The disruption of the hydrogen bond network at the head of ceramides by terpenes is a probable mechanism for enhanced permeation of IMH, as supported by the FT-IR results of this study and literature reports on DSC and ESR. It was observed that the hydrogen bond accepting and donating strength along with selfassociation of terpene molecules is a governing factor in enhanced permeation of IMH by terpenes. Better insight into the terpene action on the lipid bilayer head and speculation of H-bond breaking between head groups can be gained effectively by FT-IR through the use of a model lipid (ceramide, fatty acid and cholesterol) membrane, where interference of protein will be minimized.
Acknowledgements One of the authors (AKJ) is thankful to Council of Scientific and Industrial Research, India (CSIR,
Grant sanction no. 01(1460) / 97 / EMRII) for financial support.
References [1] W. Hammer, F. Sjoqvist, Plasma levels of monomethylated tricyclic antidepressants during treatment with imipraminelike compounds, Life Sci. 6 (1967) 1895–1903. [2] G.W. Cleary, Topical delivery systems: A medical rationale, in: V.P. Shah, H.I. Maibach (Eds.), Topical Drug Bioavailability, Bioequivalence and Penetration, Plenum Press, New York, 1993, pp. 17–68. [3] R. Panchagnula, Transdermal drug delivery of tricyclic antidepressants: feasibility study, STP Pharm. Sci. 6 (1996) 441–444. [4] P.W. Stott, A.C. Williams, B.W. Barry, Characterization of complex coacervates of some tricyclic antidepressants and evaluation of their potential for enhancing transdermal flux, J. Control. Release 41 (1996) 215–227. [5] D.L.J. Opdyke, Monographs on fragrance raw materials, Food Cosmet. Toxicol. 11–17 (Suppl.) (1973–1979). [6] A.C. Williams, B.W. Barry, Terpenes and the lipid-proteinpartitioning theory of skin penetration enhancement, Pharm. Res. 8 (1991) 17–24. [7] K. Zaho, J. Singh, In vitro percutaneous absorption enhancement of propranolol hydrochloride through porcine epidermis by terpene / ethanol, J. Control. Release 62 (1999) 359–366. [8] Y. Akithoshi, K. Takayama, Y. Machida, T. Nagai, Effect of cycohexanone derivatives on percutaneous absorption of ketoprofen and indomethacin, Drug Design Deliv. 2 (1988) 239–345. [9] H.R. Moghimi, A.C. Williams, B.W. Barry, A lamellar matrix model for stratum corneum intercellular lipids IV. Effects of terpene penetration enhancers on the permeation of 5-fluorouracil and oestradiol through the matrix, Int. J. Pharmacol. 145 (1996) 49–59. [10] R.C. Scott, M. Walker, P.H. Dugard, In vitro percutaneous absorption experiments. A technique for production of intact epidermal membrane from rat skin, J. Soc. Cosmet. Chem. 37 (1986) 35–41. [11] J.E. Harrison, A.C. Watkinson, D.M. Green, J. Hadgraft, K. Brain, The relative effect of Azone and Transcutol on permeant diffusivity and solubility in human stratum corneum, Pharm. Res. 13 (1996) 542–546. [12] D.J. Moore, M.E. Rerek, Insights into the molecular organization of lipids in the skin barrier from infrared spectroscopy studies of stratum corneum lipid models, Acta Dermatol. Venereol. Suppl. 208 (2000) 16–22. [13] Y.C. Martin, Calculation of the physical properties of compounds, in: Y.C. Martin (Ed.), Quantitative Drug Design, Marcel Dekker, New York, 1978, pp. 61–110. [14] A. Martin, P. Bustamante, A.H.C. Chun, State of matter, in: Physical Pharmacy, B.I. Waverly, New Delhi, 1996, pp. 22–52. [15] M.A. Yamne, A.C. Williams, B.W. Barry, Terpene penetra-
A.K. Jain et al. / Journal of Controlled Release 79 (2002) 93 – 101 tion enhancers in propylene glycol / water co-solvent systems: effectiveness and mechanism of action, J. Pharm. Pharmacol. 47 (1995) 978–989. [16] B.W. Barry, Mode of action of penetration enhancers in human skin, J. Control. Release 6 (1987) 85–97. [17] G.M. Golden, J.E. McKie, R.O. Potts, Role of stratum corneum lipid fluidity in transdermal drug flux, J. Pharm. Sci. 76 (1987) 25–28. [18] P.A. Cornwell, B.W. Barry, J.A. Bouwstra, G.S. Gooris, Modes of action of terpene penetration enhancers in human skin; differential scanning calorimetry, small-angle X-ray
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diffraction and enhancer uptake studies, Int. J. Pharm. 127 (1996) 9–26. [19] S. Kitagawa, A. Hosokai, Y. Kaseda, N. Yamamoto, Y. Kaneko, E. Matsuoka, Permeability of benzoic acid derivatives in excised guinea pig dorsal skin and effects of Lmenthol, Int. J. Pharm. 161 (1998) 115–122. [20] H.R. Moghimi, A.C. Williams, B.W. Barry, A lamellar matrix model for stratum corneum intercellular lipids. V. Effects of terpene penetration enhancers on the structure and thermal behaviour of the matrix, Int. J. Pharm. 146 (1997) 41–54.
Transdermal drug delivery of imipramine hydrochloride. I. Effect of terpenes Amit Kumar Jain, Narisetty Sunil Thomas, Ramesh Panchagnula* Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research ( NIPER), Sector 67, Phase-X, Mohali-160062, Punjab, India Received 14 February 2001; accepted 17 October 2001
Abstract The objective of this investigation was to study the effect of different terpenes on IMH permeation in EtOH:W (2:1) system. Permeation studies of IMH were carried out with unjacketed Franz diffusion cells through rat skin. The flux of IMH with terpenes was found to be significantly higher than that in control (EtOH:W, 2:1) (P,0.05). Amongst all studied terpenes, menthol, terpineol, cineole and menthone were found to be effective permeation enhancers for IMH. It was found that the contribution of diffusivity in enhanced permeation of IMH was much higher in comparison to partitioning of IMH in skin with terpene treatment. Results of this study were explained with the help of H-bond breaking potential and self-association of terpenes. In order to elucidate the effect of terpenes on stratum corneum barrier FT-IR was used. 2002 Elsevier Science B.V. All rights reserved. Keywords: Permeation enhancer; Terpene; FT-IR; Imipramine hydrochloride
1. Introduction TCAs are widely used in treatment of unipolar and bipolar depression, which is characterized by extreme sadness, despair and anhedonia. The problem of high first-pass metabolism, variation in gastroinAbbreviations: DCR, diffusion coefficient ratio; ER, enhancement ratio; EtOH, ethanol; FT-IR, Fourier-transform infrared spectroscopy; IMH, imipramine hydrochloride; PCR, partition coefficient ratio; SC, stratum corneum; SNK, Student–Newman– Keuls; TCAs, tricyclic antidepressants; TDDS, transdermal drug delivery system; W, water *Corresponding author. Tel.: 191-172-214-682 / 688; fax: 191-172-214-692. E-mail address: [email protected] (R. Panchagnula).
testinal absorption [1] and patient non-compliance with conventional per oral delivery can be overcome with transdermal delivery of TCAs. As transdermal delivery offer inherent advantages such as: (a) bypasses first pass metabolism; (b) enables control of input; (c) avoids problems of stomach emptying, pH effects and enzymatic deactivation associated with gastrointestinal tract passage [2]. However, their poor permeability and high dose are the major hurdles in delivery across the skin. Panchagnula [3] and Stott et al. [4] have suggested the use of solvent, e.g., dimethylsulfoxide, dimethylacetamide and complex coacervation technique, respectively, for enhancement of TCAs permeation through skin [3,4]. With these approaches flux of IMH was not high
0168-3659 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00524-7
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A.K. Jain et al. / Journal of Controlled Release 79 (2002) 93 – 101
enough to achieve the desired plasma level. Therefore, in this investigation terpenes were selected as permeation enhancers to study their effect on IMH in vitro permeation across the rat skin. Terpenes are non-toxic and non-irritant [5] to skin and are extensively used in transdermal delivery as permeation enhancers with hydrophilic (5-fluorouracil [6], propanolol hydrochloride [7]) and lipophilic (indomethacin and ketoprofen [8], estradiol [9]) drugs. In addition, FT-IR was used to elucidate the mechanism of action of terpenes effect on lipid bilayer of SC.
2. Materials IMH and cineole were procured from Sigma (St. Louis, MO, USA). [ 3 H]IMH (specific activity, 48.00 Ci / mmol) was obtained from Du Pont (Wilmington, DE). Pulegone, a-terpineol, menthol, ethanol, carvone and menthone were purchased either from Merck, (Germany) or Fluka Chem (Germany). Tissue solubilizer (NCS II) and scintillation cocktail (BCS 104) were purchased from Amersham (UK). All other chemicals used were of reagent grade.
3. Methods
3.1. Preparation of full thickness skin The skin samples were harvested from the dorsal surface of the Sprague–Dawley rats. Hair on dorsal skin of animal was removed with animal hair clipper (Aesculp, Germany), subcutaneous tissue was surgically removed and dermis side was wiped with isopropyl alcohol to remove residual adhering fat. The skin was washed with distilled water, wrapped in aluminium foil and stored in a freezer at 2208C till further use. The protocol for this study was approved by NIPER’s Institutional Animal Ethics Committee.
3.2. Separation of epidermis from full thickness skin Frozen skin was thawed at room temperature and treated with 2 M sodium bromide solution in water
for 6 h [10]. The epidermis was separated by using a cotton swab moistened with distilled water. Epidermal sheet was cleaned by washing with distilled water and dried under vacuum. Dried epidermal sheets were stored in desiccator until further use.
3.3. Ex vivo permeation study IMH permeation across the skin was determined (n54) using unjacketed Franz cells with a diffusional surface area of 0.785 cm 2 . The cells were placed on dry heater and stirrer block supplied by Permegear (USA). Phosphate buffer (pH 7.4), containing 0.01% (w / v) sodium azide as a preservative was used as the receptor medium. The receptor fluid was sonicated to remove dissolved gases and equilibrated at 378C before placing in the receptor compartment. Cells were filled with receptor fluid and skin was mounted on diffusion cell with dermal side in contact with the receptor medium, then equilibrated at 378C for 8–10 h. IMH solution (200 ml of 75 mg / ml and spiked with 1 mCi / ml) in solvent system, i.e., EtOH:W (2:1) with or without terpene (5%, w / v) was applied to SC side in the donor compartment. Donor compartment was covered with paraffin film. After application of drug solution, samples (200 ml) were withdrawn from the receptor compartment at different time intervals and analysed for drug content by radiochemical analysis. Sample volume was immediately replaced with fresh receptor medium after each sampling.
3.4. Determination of partition coefficient Partition coefficient of IMH at 32618C between EtOH:W (2:1) system with or without terpene and epidermal sheet were determined in triplicate. Epidermal sheets were cut into 1-cm 2 pieces and each piece was dipped into separate screw cap vials containing 1 ml of water with 0.01% (w / v) sodium azide, for 36 h. Epidermal sheets were then blotted dry, weighed and soaked into 1 ml of various solvent systems containing IMH (1.16 mg / ml with 0.016 mCi / ml). All vials were kept under constant shaking in water bath (Julabo, Germany) at 328C for 12 h. Epidermal sheets were then blotted dry, transferred to glass scintillation vials and solubilized in 600 ml of tissue solubilizer. Five ml of scintillation cocktail
A.K. Jain et al. / Journal of Controlled Release 79 (2002) 93 – 101
and 100 ml of glacial acetic acid were added into each glass scintillation vial [6]. After 12 h, vials were counted for radioactivity in liquid scintillation counter (Wallac 1405, Finland). Radioactivity left in the solvents in which epidermal sheets were soaked was also counted in duplicate for mass balance.
3.5. FT-IR Dried epidermal sheet was cut into approximately 2-cm 2 pieces and soaked in water containing 0.01% of sodium azide for 36 h. Epidermal pieces were dried under ambient condition for overnight and then kept under vacuum in phosphorus pentoxide (P2 O 5 ) desiccators for 15 min to ensure complete removal of free water. FT-IR spectra of each epidermal piece were recorded before terpene treatment (control) and immediately after this, pieces were dipped into respective terpene solutions (5%, w / v) in EtOH:W (2:1) system for 12 h. Each piece after treatment was washed three times with EtOH:W (1:1) and water, then air dried for 2 h. Samples were kept under vacuum in phosphorus pentoxide (P2 O 5 ) desiccators for 15 min to remove alcohol and free terpenes completely. FT-IR spectra of all pieces treated with terpenes were recorded on Nicolet (USA) FT-IR spectrophotometer with following acquisition parameters; resolution of 2 cm 21 , number of scans was 100 and detector was DTGS. All epidermal pieces after drying were kept under vacuum for 36 h and FT-IR spectra were recorded again to ensure that terpenes remained on surface after treatment were removed completely by washing and were not interfering with skin lipid and protein peaks.
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SSF Permeability 5 ]] Cd where, SSF is steady-state flux (mg cm 22 h 21 ), Cd is concentration in donor compartment. Since there is a possibility of unpredictable alteration in diffusion pathlength (h) after terpene treatment, therefore, in order for better approximation of diffusivity (D) of IMH, D/h 2 was derived from lag time by following equation [11]; D/h 2 5 1 /(6 ? t lag ) where, t lag is lag time in hours.
4. Results and discussion Permeation profile of IMH with and without the treatment of terpenes is shown in Fig. 1. Flux values of IMH, with and without terpene treatment are given in Table 1, in decreasing order is as follows; menthol$cineole.terpineol.menthone. pulegone.carvone.control (EtOH:W, 2:1). Flux of IMH in terpenes was significantly different from control (EtOH:W, 2:1) (P,0.05). However, among the terpenes, flux of IMH with menthol, cineole and
3.6. Data analysis Cartesian plots with cumulative amount of drug present in receptor compartment versus time were plotted. Flux values (mg cm 22 h 21 ) were calculated from the slopes of the steady-state portion of the plots. Lag times were calculated from the intercepts of extrapolated steady-state flux to the x-axis. Permeability values were calculated by the following equation;
Fig. 1. Effect of different class of terpenes on IMH permeation profile.
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Table 1 Permeation parameters of IMH with and without terpene treatment, mean (6S.D.) Chemical class Control Alcohol Ether Ketone
E:W (2:1) Menthol Terpeneol Cineole Menthone Pulegone Carvone
J (mg cm 22 h 21 )
Lag time (h)
Kp 3(10 3 ) (cm/h)
P
45.7 (18.80) 754.0* (159.60) 530.0* (188.00) 623.0* (97.80) 452.2* (90.50) 231.5* (40.80) 288.7* (30.50)
32.29 (13.52) 5.81* (0.82) 6.54* (2.38) 5.43* (2.36) 4.69* (1.19) 9.78* (1.39) 11.46* (1.87)
0.61 10.50 7.06 8.03 6.02 3.08 3.84
2.04 1.90 2.70 3.19 1.91 2.73 1.84
(0.62) (0.29) (0.44) (0.45) (0.13) (1.53) (0.20)’
D/h 2 (h 21 )
PCR
DCR
ER
0.0059 (0.002) 0.0290 (0.003) 0.0285 (0.011) 0.0392 (0.026) 0.0378 (0.012) 0.0151 (0.002) 0.0172 (0.002)
– 0.93 (0.14) 1.32 (0.21) 1.56 (0.22) 0.93 (0.06) 1.34 (0.75) 0.90 (0.09)
– 4.92 (0.63) 4.83 (1.93) 6.65 (4.54) 6.42 (2.14) 2.56 (0.44) 2.93 (0.42)
– 16.69 11.58 13.61 9.80 5.03 6.27
*Significantly different from control, P,0.05. PCR, partition coefficient with terpene treatment / partition coefficient without terpene treatment; DCR, diffusivity with terpene treatment / diffusivity without terpene treatment; and ER, flux with terpene treatment / flux without terpene treatment.
terpineol was significantly different from pulegone and carvone (P,0.05), whereas flux of IMH in menthol and cineole were comparable. Lag time of IMH with terpene treatment was significantly reduced as compared to control (EtOH:W, 2:1) (P,0.05) and order of terpene effect on lag time in decreasing order is as follows control.carvone.pulegone.terpineol.menthol. cineole.menthone. The main barrier or rate-limiting step in transdermal drug delivery of polar, water-soluble drug such as IMH is the lipophilic part of SC, in which lipids (ceramides) are arranged in a bilayer. Ceramides (especially ceramide 2 and ceramide 5, which are abundantly present in SC) are tightly packed in the bilayer due to the high degree of hydrogen bonding. The amide I group of ceramide is hydrogen bonded to amide I group of another ceramide and a tight network of hydrogen bonding is formed at the head of ceramides. This hydrogen bonding provides strength and stability to lipid bilayer and imparts the barrier property to SC. Recently, it has been proved with model SC lipids (hexadecanoic acid, cholesterol, bovine non-hydroxy fatty acid ceramide and bovine hydroxy fatty acid ceramide) that amide I of ceramide 5 is laterally hydrogen bonded between head of ceramides [12]. In addition, amide I of ceramide 2 is responsible for a high degree of lateral and transverse hydrogen bonding, which involves amide I group and carbonyl moiety of opposite ceramide and fatty acid, respectively. When skin is treated with terpenes, the existing network of hydrogen bonds between ceramides may get loosened
because of ‘competitive hydrogen bonding’ (Fig. 2). The hydrogen bond network at the head of ceramides breaks as terpenes (e.g., menthol, terpineol) enter into the lipid bilayer of SC, since alcoholic –OH group is more electronegative than –NH of amide I [13]. Since alcoholic –OH group can accept or donate the hydrogen bond, it leads to disruption of existing hydrogen bonding between ceramides head groups, thereby facilitating the permeation of IMH and is also reflected by high ER with menthol and terpineol (Table 1). However, in case of menthone, pulegone, carvone and cineole, only hydrogen bond accepting moieties, viz. carbonyl, and ether groups are present, respectively. The extent of disruption of the hydrogen bond network between the ceramides heads is less, as is evident from relatively less ER with pulegone and carvone (Table 1). With cineole and menthone, ER and DCR were found to be higher than that of pulegone and carvone. Of all terpenes studied, the boiling point (bp) of cineole (bp 1738C) and menthone (2108C) is approximately 408C and 2–178C lesser than that of other terpenes, respectively, including menthol (bp 2158C) and terpineol (bp 2178C). This is an indication of weak cohesive or self-association of cineole and menthone molecules [14] or in other words oxygen of functional ether and carbonyl group is in free form for interaction. Therefore, the energy required for competitive hydrogen bonding in skin ceramides is relatively less for cineole and menthone, which can be attributed to the higher ER and DCR as found with these terpenes. On the other hand, in case of menthol,
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Fig. 2. Mechanistic aspects of terpenes action on lipid bilayer of SC.
terpineol, pulegone and carvone, additional energy is required to free the respective functional group from strong self-association, as reflected by higher boiling point (Table 2). Hence low DCR was found with menthol and terpineol. Whereas in case of pulegone and carvone self-association and presence of only hydrogen bond acceptor is responsible for weak interaction with skin ceramides, lower ER and DCR were found as compared to other terpenes. Menthone, pulegone and carvone belong to same class of terpene, i.e., ketone terpene (carbonyl group), but menthone was found to be an effective permeation enhancer for IMH when compared to the other two ketone terpenes. This difference in effectiveness of ketone terpenes as permeation enhancers may be because of different degrees of unsaturation in Table 2 Boiling point of terpenes Terpene
Boiling point (8C)a
Menthol Cineole Terpineol Menthone Pulegone Carvone
215 173 217 210 224 230
a
Boiling points of terpenes are taken from Merck and Aldrich catalogues.
ketone terpenes, e.g., menthone (0), pulegone (2) and carvone (3); (degree of unsaturation), resulting in difference in cohesiveness as is also indicated by their boiling point (Table 2). Partition coefficients of IMH in epidermis / vehicle (EtOH:W (2:1) with or without terpene) system were determined and results are shown in Table 1. Results of this study indicate that the contribution of partitioning in enhanced permeation of IMH after terpene treatment is negligible, as shown by lower PCR values, and are in agreement with previous finding [6,15]. FT-IR of rat epidermis after 12 h treatment shows no shift in wavenumber (Fig. 3a) and changes produced in peak height (2850 and 2920 cm 21 ) and area (2850 and 2920 cm 21 ) does not significantly differ from changes produced by the EtOH:W (2:1) system (results not shown). The EtOH:W (2:1) system with or without terpenes was found to be inefficient to extract the lipid from rat epidermis, as is evident from presence of –C5O peak of ester at 1740 cm 21 region (Fig. 3b). As discussed earlier ceramide 2 and 5 form H-bonds between each other in the SC lipid bilayer and ceramide 2 is responsible for transverse H-bonding in the lipid bilayer, thus causing the split in peak at 1650 cm 21 (amide I) which was found in model membrane prepared from SC lipids [12]. A similar split in the peak at 1650 cm 21 region was observed in our control experi-
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Fig. 3. FT-IR spectra of rat epidermis. (A) After 12 h treatment with (a) water, (b) EtOH:W (2:1), (c) cineole, (d) menthol, (e) terpineol, (f) menthone, (g) pulegone, (h) carvone. (B) Individual control before treatment with different terpenes (a) EtOH:W (2:1), (b) cineole, (c) menthol, (d) terpineol, (e) menthone, (f) pulegone, (g) carvone. (C) After 12 h treatment with different class of terpenes.
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Fig. 3. (continued)
ments also (Fig. 3b). After treatment with terpenes, only a single peak at 1650 cm 21 was found (Fig. 3c), which indicates breaking of H-bonds by terpenes. However, the possibility of breaking of H-bond network existing in a-keratin and thereby expansion of corneocytes cannot be overlooked for the EtOH:W (2:1) system because the split at 1650 cm 21 also disappeared in this system. Since the main barrier in permeation is the lipid bilayer, this effect may not be significant, unless the lipid bilayer fluidity increases [16] as is indicated by lower flux values with the EtOH:W (2:1) system. With ethanol, bilayer fluidity is not increased as found by DSC studies from literature and there was no shift in lipid transition temperature after ethanol treatment [17]. However, with terpenes in the EtOH:W (2:1) system, apart from the EtOH:W effect on a-keratin structure, increased lipid bilayer fluidity by terpenes may also be responsible for higher ER. Since it was observed that the FT-IR could not differentiate the effect of EtOH:W (2:1) and terpenes on protein and lipid bilayer of epidermis, respectively, therefore DSC and
ESR data from literature was used to support the assumption of breaking of H-bonds between the head group of ceramides on treatment with terpenes. With DSC, It has been reported that cineole and menthone induce shifts in transition temperature along with increased entropy (measure of disorder) at physiological temperature or below transition temperature [15]. This is attributed to loosening of highly organised lipid bilayer structure due to breaking of the H-bond network. The hypothesis of breaking or loosening of the H-bond network between ceramides head groups by –OH or –C=O group of terpenes can also be supplemented by the effect of limonene (terpene without –OH or –C=O group) on the entropy of lipid transition. Though it has been reported that limonene caused a shift in transition temperature of skin, entropy associated with lipid transition was found to be unchanged [15,18]. In other words, limonene is not able to break the Hbond network between ceramides due to absence of a hydrogen bond accepting or donating group. It has been reported that ER for 5-fluorouracil (a polar,
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hydrophilic) with limonene was less than that of cineole, menthone and nerolidol. With ESR, it has been reported that the fluidity of the lipid bilayer is increased after menthol treatment [19]. It has been reported that cineole forms a pool in model SC matrix and enhances the permeation of 5-fluorouracil and oestradiol [20]. At 5% (w / v) concentration, no characteristic peak of terpene was found in FT-IR spectra of rat epidermis after 12 h treatment with terpene, which implies that at this concentration terpenes may not form a pool inside the lipid bilayer of SC. As described in this study, permeation of a watersoluble, polar drug such as IMH can be increased significantly through breaking of hydrogen bonds between ceramide heads by terpenes with weak selfassociation or which have the ability of donating or accepting H-bond.
5. Conclusions Amongst all the terpenes, menthol was found to be an effective permeation enhancer for IMH. In general, it was observed that terpenes with minimum degree of unsaturation, like menthol, cineole, menthone, are good permeation enhancers for polar and water-soluble drugs, such as IMH. The disruption of the hydrogen bond network at the head of ceramides by terpenes is a probable mechanism for enhanced permeation of IMH, as supported by the FT-IR results of this study and literature reports on DSC and ESR. It was observed that the hydrogen bond accepting and donating strength along with selfassociation of terpene molecules is a governing factor in enhanced permeation of IMH by terpenes. Better insight into the terpene action on the lipid bilayer head and speculation of H-bond breaking between head groups can be gained effectively by FT-IR through the use of a model lipid (ceramide, fatty acid and cholesterol) membrane, where interference of protein will be minimized.
Acknowledgements One of the authors (AKJ) is thankful to Council of Scientific and Industrial Research, India (CSIR,
Grant sanction no. 01(1460) / 97 / EMRII) for financial support.
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