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Electrically-assisted transdermal delivery of buprenorphine
Journal of Controlled Release 73 (2001) 197–203 www.elsevier.com / locate / jconrel
Electrically-assisted transdermal delivery of buprenorphine Sagarika Bose a , William R. Ravis a , Yuh-Jing Lin a , Lei Zhang b , ¨ Gunter A. Hofmann b , Ajay K. Banga c , * a
Department of Pharmacal Sciences, School of Pharmacy, Auburn University, Auburn, AL 36849 -5503, USA b Genetronics, Inc., 11199 Sorrento Valley Road, San Diego, CA 92121 -1334, USA c Department of Pharmaceutical Sciences, School of Pharmacy, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341 -4155, USA Received 24 July 2000; accepted 20 January 2001
Abstract The objective of this study was to explore the electrically assisted transdermal delivery of buprenorphine. Oral delivery of buprenorphine, a synthetic opiate analgesic, is less efficient due to low absorption and large first-pass metabolism. While transdermal delivery of buprenorphine is expected to avoid the first-pass effect and thereby be more bioavailable, use of electrical enhancement techniques (iontophoresis and / or electroporation) could provide better programmability. Another use of buprenorphine is for opiate addiction therapy. However, a patch type device is subject to potential abuse as it could be removed by the addict. This abuse can be prevented if drug particles are embedded in the skin. The feasibility of doing so was investigated by electro-incorporation. Buprenorphine HCl (1 mg / ml) in citrate buffer (pH 4.0) was delivered in vitro across human epidermis via iontophoresis using a current density of 0.5 mA / cm 2 and silver–silver chloride electrodes. Electroporation pulses were also applied in some experiments. For electro-incorporation, drug microspheres or particles were driven into full thickness human skin by electroporation. It was observed that the passive transdermal flux of buprenorphine HCl was significantly enhanced by iontophoresis under anodic polarity. The effectiveness of electro-incorporation seemed inconclusive, with pressure also playing a potential role. Delivery was observed with electro-incorporation, but the results were statistically not different from the corresponding controls. 2001 Elsevier Science B.V. All rights reserved. Keywords: Iontophoresis; Electroporation; Buprenorphine; Skin transport
1. Introduction Buprenorphine is a narcotic analgesic which is approximately 30–50 times more potent than morphine. It is commercially available (Buprenex ; Reckitt and Colman, VA) for relief of moderate to *Corresponding author. Tel.: 11-678-547-6243; fax: 11-678547-6423. E-mail address: banga [email protected] (A.K. Banga). ]
severe pain. It is administered parenterally by intramuscular or slow intravenous injection at a typical dose of 0.3–0.6 mg every 6–8 h. The patient may have to take the injection 3–4 times a day, depending on the severity of the pain. Thus, controlled release parenterals or alternate delivery systems are desirable. Oral delivery is impractical as buprenorphine undergoes an extensive first pass metabolism. Buccal or oral mucosal delivery of buprenorphine has been investigated [1,2] and a sublingual system
0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00298-X
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S. Bose et al. / Journal of Controlled Release 73 (2001) 197 – 203
has been commercialized in Europe. Nearly 75% of buprenorphine was shown to release from a bioadhesive Carbopol -based buccal patch following a 24 h incubation in phosphate buffer [1]. Transdermal delivery offers a viable alternative [3–6]. A prodrug strategy has been used with some success where the drug is metabolized to the parent compound while in the skin [5]. However, it has been shown that transdermal delivery of buprenorphine hydrochloride by itself can also result in adequate plasma levels for sustained analgesic effect. There was a delay in onset which could be reduced by using an ethanolic solution [6]. Electrically-modulated delivery [7] by iontophoresis and / or electroporation will provide a further advantage of programming the dose in proportion to the level of analgesia desired. Buprenorphine can also be used for opiate addiction therapy [8] as it has lower abuse potential and milder withdrawal symptoms compared to methadone. For this use, a patch type device may not be desirable as it is subject to potential abuse since it could be easily removed by the addict. If buprenorphine drug particles can be successfully incorporated into the skin, then the drug may release from the formulation for an extended period of time, and the advantage of this approach would be that the formulation cannot be easily removed by the patient / addict. Buprenorphine is a very lipophilic drug. Its hydrochloride salt still remains lipophilic despite the small hydrophilic addition (HCl) which is not able to shield the bulky hydrophobic moiety. However, the lipophilicity is reduced for the HCl salt which is able to better permeate the skin than the free base as it can partition out of the epidermis into the dermis more easily due to its moderate lipophilicity [3]. There is one report in the literature on the iontophoretic delivery of buprenorphine to weanling Yorkshire swine [9]. Though therapeutic levels were achieved, the flux was very low and a strong skin depot was observed. This could be because the drug is very lipophilic. The use of electroporation alone or in combination with iontophoresis to deliver buprenorphine has not been investigated. Electroporation directly enhances the permeability of the stratum corneum, the outermost resistive layer of the skin, by creating new pathways in addition to existing ones.
2. Materials and methods
2.1. Materials Buprenorphine HCl (MW 504.11) and 3 H labeled buprenorphine was obtained from RBI (Natick, MA) and Sigma (St. Louis, MO), respectively. Sodium chloride, sodium phosphate monobasic and dibasic citric acids were obtained from Fisher Scientific (Pittsburgh, PA). Scintillation cocktail (ULTIMAGOLDE) and Solvable tissue and gel solubilizer was purchased from Packard (Meriden, CT). Silversilver chloride electrodes were obtained from In Vivo Metric (Healdsburg, CA). Silver wires were obtained from Aldrich Chemical Co. (Milwaukee, WI). Human cadaver skin was obtained from the Cooperative Human Tissue Network (Birmingham, AL) and was frozen within 12 h of death and supplied as full thickness skin. Pig skin was obtained from Auburn University, College of Veterinary Medicine (Auburn, AL). Neonatal pigs were used (Genus species: Sus scrofa) to procure skin as these were readily available. All the skins were frozen at 2808C and thawed just before use.
2.2. Preparation of skin For full-thickness skin (human and pig), the abdominal skin was thawed before use by immersing it in distilled water at room temperature. The underlying subcutaneous fat was gently scraped off till the skin was about 1–1.5 mm thick. Then it was cut into appropriately sized pieces and mounted on Franz transdermal diffusion cells. For removal of epidermis from full-thickness human skin, the skin was heated in water at 608C for 45 s and then rubbed gently by two spatulas. The epidermal membrane was teased off from the underlying dermis by forceps. Then, it was spread over a piece of parafilm so that it becomes easy to cut into appropriate pieces.
2.3. Transdermal studies Franz (vertical) diffusion cells were used for in vitro permeation studies. The donor half was exposed to room temperature (258C), while the receptor half was maintained at 378C. The skin was
S. Bose et al. / Journal of Controlled Release 73 (2001) 197 – 203
thawed and mounted on the diffusion cells. The receptor phase was filled with 5.0 ml of pH 6.0 phosphate buffer (as buprenorphine is soluble at this pH). The donor phase (0.5 ml) was made up of cold buprenorphine (1 mg / ml) spiked with tritiated buprenorphine in pH 4.0 citrate buffer. Samples (0.5 ml) were taken from the receptor phase and analyzed by LSC. Experiments were performed in triplicate and all data are plotted as mean6standard deviation. For iontophoresis, silver–silver chloride electrodes were used because they do not cause electrolysis of water, which may result in pH shifts. Silver wire was used as the anode and silver–silver chloride was used as the cathode. The anode was placed in the donor solution and the cathode was placed in the receptor unless otherwise specified. A current of 0.5 mA / cm 2 was applied for 4 h and sampling was continued for 24 h. For Electroporation, platinum wires were used as electrodes (both cathode and anode), and the exponential pulse generator was a BTX Electro Cell Manipulator Model 600 (Genetronics Inc., San Diego, CA). High voltage (.100 V) pulses (10 or 20 ms) were applied to the skin. The pulse rate or the number of pulses per minute was manually controlled as the protocols of the experiments were changed. Anode was placed in donor chamber and cathode was placed in receptor. In some experiments, the combined use of iontophoresis and electroporation was explored.
2.4. Electro-incorporation studies Electro-incorporation [10,11] studies were performed using either microspheres or drug particles (solid state) placed on full thickness human skin. Buprenorphine microspheres were prepared by the method of Ravis et al. [12] by solvent evaporation methods, using PLGA (65:35) as the biodegradable polymer. Briefly, Buprenorphine HCl was dissolved in methylene chloride with PLGA polymer. The solution was added to purified water and following mixing by vortexing, the dispersion was added to a solution of poly(vinyl alcohol) and mixed for 3 h. After centrifuging, the microspheres were washed with water. Buprenorphine was labeled with 3 Hbuprenorphine in the microspheres. For preparation
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of drug particles, buprenorphine was dissolved in ethanol and tritiated buprenorphine (in ethanolic solution) was added to this. The ethanol was then evaporated under nitrogen to get an intimate mixture of cold and hot buprenorphine. This was suspended in pH 7.4 phosphate buffer (1 mg / 6 ml of suspension), as buprenorphine is practically insoluble at this pH. The microsphere or drug particles’ suspension (6 ml) was placed on skin and covered with a meander electrode. A meander electrode consists of an interweaving array of metal fingers coated on a thin plastic film which can be placed on the skin. Electroporation pulses were applied via these metal fingers on the meander electrode. This creates an electric field which breaks down the stratum corneum by a yet unknown mechanism. The electrode is in direct contact with the skin here and low voltage is given so that it does not damage the skin. A slight pressure was maintained on the electrode during pulsing by clamping the whole set-up on the upper half of a Franz cell. The drug suspension under the meander electrode substitutes for the donor solution and the upper half of the Franz cell is being used merely to hold the meander electrode in place. The skin was covered with parafilm and a metal plate before clamping to maintain some pressure on it. Meander electrode was connected to the electroporation equipment and pulses were given. After pulsing, the skin was washed several times with pH 4.0 citrate buffer to remove any drug adhering to the surface as buprenorphine is more soluble at this pH. The skin was then stripped five times with tape. The remaining skin was digested with Solvable , a tissue and gel solubilizer at 708C until the skin had completely dissolved. The residual radioactivity in the skin and strips was then assayed separately by liquid scintillation counter (LSC).
2.5. Skin impedance studies The electrical properties of the skin in the passive state were measured using a function generator (V0 5 1V; repetition at f51 KHz; sine wave). The resistance of the chamber without the skin, R Bulk , was also measured in a similar fashion. The skin was pulsed using the BTX pulser. To measure the applied
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voltage (V0 ), a digital oscilloscope was used and the trace stored in channel 1. The voltage developed across the 15 V resistor in series with the chamber was measured at channel 2 (Vs ). This would give the total current through the chamber. The effective transdermal voltage (VSkin ) was calculated and was much lower than the applied voltage across electrodes (Velectrode ), a typical VSkin being about 50 V for an applied Velectrode of 500 V. The voltages are given as Velectrode in this paper.
3. Results and discussion
3.1. Transdermal transport studies The effect of formulation pH on iontophoretic transport of buprenorphine was investigated. With the pH of donor solution at 4.0, the average cumulative amount in the receptor was 15.8961.94 mg / cm 2 at 24 h while it was only 5.161.8 mg / cm 2 when the donor pH was 5.0. This may relate to the change in the charge of the skin (and hence electroosmotic flow) at different pH or to the higher solubility of buprenorphine at pH 4.0. Fig. 1 shows the average cumulative amount present in the receptor during anodic (anode in donor) and cathodic (cathode in donor) delivery over 24 h following a 4-h current application. The amount delivered under anode (15.8961.94 mg / cm 2 ) was much higher than that delivered under cathode (1.9060.06 mg / cm 2 ). The results are statistically significant (P,0.05). Passive delivery (no current) resulted in very low permeation, similar to cathodic delivery. Buprenorphine has a positive charge at pH 4.0 and this study establishes that it should be delivered under anode. The permeation of buprenorphine seems to continue even after stopping the current. This could be due to a skin depot (reservoir) which is slowly released into the receptor. Alternatively, the skin may have been compromised by iontophoresis so that the drug is diffusing from the donor to the receptor. The latter is not expected at the low current density used. However, to investigate which one of these is most likely explanation, a desorption study was done in which drug solution in donor is replaced with buffer after 4 h of iontophoresis and then sampling was continued to analyze the drug being released from the depot
Fig. 1. Comparison between anodic, cathodic, and passive transport of buprenorphine after 4 h of iontophoresis (0.5 mA / cm 2 ) followed by passive delivery upto 24 h through full thickness pig skin (n53).
(desorption). Desorption was carried out under passive (no current after 4 h) or iontophoresis (anodic iontophoresis reapplied after replacing drug solution with plain buffer) conditions (Fig. 2). The data shows that buprenorphine was released into receptor even after donor solution was replaced with buffer, suggesting the presence of a skin depot. The difference between passive and iontophoretic desorption was not statistically significant (P,0.05), suggesting that the skin reservoir of lipophilic buprenorphine cannot be pushed out by the iontophoretic current used in these experiments. Fig. 3 shows the difference between iontophoresis, iontophoresis coupled with electroporation, and electroporation alone using full thickness pig skin. All the results are statistically significant (P,0.05). The electroporation protocol was 20 pulses (10 ms each) of 500 V (applied voltage) at time zero while iontophoresis current was applied for 4 h. Electroporation alone was unable to enhance transport perhaps because there is no sufficient driving force (as in iontophoresis) to push the drug into the skin permeabilized by electroporation. In addition, the total amount of charge delivered by
S. Bose et al. / Journal of Controlled Release 73 (2001) 197 – 203
Fig. 2. Desorption of buprenorphine reservoir in skin under passive or iontophoretic conditions following 4 h (0.5 mA / cm 2 ) of iontophoretic delivery of buprenorphine (n53).
Fig. 3. Effect of iontophoresis for 4 h (0.5 mA / cm 2 ) and electroporation (applied voltage 500 V, 10 ms, 20 pulses) followed by passive delivery for 24 h on buprenorphine delivery through full thickness pig skin (n53).
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electroporation is significantly less than that delivered by iontophoresis. A combination of the two modalities resulted in delivery which was over six times higher than that achieved by electroporation alone and about twice of that achieved by iontophoresis alone. In another study, a comparison between only passive transport and passive transport after electroporation was investigated using pig skin. The average cumulative amount in the receptor after 24 h for passive transport was 1.7860.69 mg / cm 2 while when 20 pulses (each of applied voltage 500 V, 10 ms) were given at time zero, the average cumulative amount in the receptor was 1.9660.41 mg / cm 2 after 24 h. Thus, electroporation only does not facilitate the transport to a significant extent. Iontophoretic delivery of buprenorphine through human epidermis is shown in Fig. 4. As can be seen, the depot effect seems less when epidermis rather than full thickness skin is used. In practical terms, this result is more meaningful since the blood circulation lies just under the epidermis and will drain most of the drug quickly, preventing the formation of a skin depot. Delivery through epidermis was also higher than
Fig. 4. Effect of iontophoresis for 4 h (0.5 mA / cm 2 ) and electroporation (applied voltage 500 V, 10 ms, 20 pulses) followed by passive delivery for 24 h on buprenorphine delivery through human epidermis (n53).
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that through full thickness skin. By extrapolating the data to typical patch sizes available on the market (about 5–30 cm 2 ), it can be seen that therapeutic doses can be easily delivered. In the case of human epidermis, electroporation failed to enhance iontophoretic delivery as used in this study. However, this could be drug and protocol specific as for calcitonin [13], electroporation did enhance iontophoretic transport across human epidermis. The impedance of the skin was measured for one protocol (applied voltage 500 V, 10 ms, 4 ppm for 30 min). The initial impedance of the skin was 6.7 kV and it fell to about 30 V during the pulses. Following electroporation, it recovered to about 1.6 kV in 24 h. The effect of number of pulses (applied voltage 500 V, 10 ms) applied was also investigated, and no difference was observed when 50 or 100 pulses were applied prior to 4 h of iontophoresis (data not shown).
3.2. Electro-incorporation studies Buprenorphine microspheres were pulsed on human full thickness skin by meander electrodes using six pulses (applied voltage 120 V, 10 ms each) at time zero. Control experiment was treated with the formulation and electrodes were placed the exact same way but no pulses were applied. The microspheres were 5–20 microns in diameter and the final product contained 7.94% drug. The electroporation applied voltage (120 V) was lower than what was used in solution studies as these electrodes are in direct contact with the skin. The results (Fig. 5) show that there was no significant difference between pulse and control experiment. In a study with a suspension of pure drug, the same amount of solid placed under meander electrode and pulsed with the same protocol as for microsphere formulation. The use of pure drug suspension resulted in significantly higher delivery. Furthermore, the experimental group had significantly higher drug than the control. A more intense protocol (20 pulses; applied voltage 120 V, 20 ms each) resulted in somewhat higher delivery but now the difference between control and experimental groups was not statistically significant (P,0.05) (Fig. 6). Considering that no pulses were given, the control values were very high in these electro-incorporation experiments. This was initially suspected due to inadequate washing protocol. How-
Fig. 5. Delivery of buprenorphine microspheres through human full thickness skin by electroincorporation (applied voltage 120 V, 10 ms, 6 pulses) (n53).
ever, careful and extensive washing did not help much to resolve the problem. Skin stripping was performed and it was observed that the control skin had significant amount of drug even after stripping. It is possible that particles sticking to the skin may
Fig. 6. Delivery of buprenorphine solid drug suspension through human full thickness skin by electroincorporation (applied voltage 120 V, 6 pulses) (n53).
S. Bose et al. / Journal of Controlled Release 73 (2001) 197 – 203
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study had relatively small effect on drug delivery. The feasibility of delivery of buprenorphine particles (microspheres or pure drug suspension) in skin by electroporation could not be established in this study with the electrical protocols used. References
Fig. 7. Amount of drug transported in 48 h following electroincorporation (applied voltage 120 V, 10 ms, 12 pulses) of buprenorphine microspheres in full thickness human skin (n53).
be driven into the skin by pressure alone. Such pressure-mediated delivery was not investigated in these experiments. Alternatively, some drug could also release from the surface of microspheres and then enter the skin. In one study, an indirect measurement of electro-incorporation was attempted by leaving the skin on Franz diffusion cells for 48 h after pulsing (12 pulses of applied voltage 120 V, 10 ms), with skin being exposed to the receptor solution. Samples were taken from the receptor and analyzed over this time period. The results (Fig. 7) show that the permeation between control and pulsed groups was statistically insignificant (P,0.05).
4. Conclusion The passive transdermal flux of buprenorphine HCl is significantly enhanced by iontophoresis. Anodic delivery was higher than cathodic delivery for a pH 4.0 formulation. A depot (reservoir) in the skin was observed following iontophoretic drug delivery. The electroporation protocols used in this
[1] J.H. Guo, Bioadhesive polymer buccal patches for buprenorphine controlled delivery: Formulation, in-vitro adhesion and release properties, Drug Dev. Ind. Pharm. 20 (1994) 2809– 2821. [2] R.L. Mcquinn, D.C. Kvam, M.J. Maser, A.L. Miller, S. Oliver, Sustained oral mucosal delivery in human volunteers of buprenorphine from a thin non-eroding mucoadhesive polymeric disk, J. Controlled Release 34 (1995) 243–250. [3] S.D. Roy, E. Roos, K. Sharma, Transdermal delivery of buprenorphine through cadaver skin, J. Pharm. Sci. 83 (2) (1994) 126–130. [4] D.A. Barrett, N. Rutter, T. Kurihara-Bergstrom, S.S. Davis, Buprenorphine permeation through premature neonatal skin, Pharm. Sci. Commun. 4 (1994) 125–130. [5] H. Imoto, Z.Q. Zhou, A.L. Stinchcomb, G.L. Flynn, Transdermal prodrug concepts: Permeation of buprenorphine and its alkyl esters through hairless mouse skin and influence of vehicles, Biol. Pharm. Bull. 19 (1996) 263–267. [6] I.R. Wilding, S.S. Davis, G.H. Rimoy, P. Rubin, T. Kuriharabergstrom, V. Tipnis, B. Berner, J. Nightingale, Pharmacokinetic evaluation of transdermal buprenorphine in man, Int. J. Pharm. 132 (1996) 81–87. [7] A.K. Banga, Electrically Assisted Transdermal and Topical Drug Delivery, Taylor and Francis, London, UK, 1998. [8] R.E. Johnson, J.H. Jaffe, P.J. Fudala, A controlled trial of buprenorphine treatment for opioid dependence, J. Am. Med. Assoc. 267 (1992) 2750–2755. [9] J. Denuzzio, K. Boericke, D. Sutter, A. Mcfarland, D. Dey, R. Cesarini, E. Monty, D. Colville, R. Bock, M. O’Connell, B. Sage, Iontophoretic delivery of buprenorphine, Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 23 (1996) 285– 286. [10] G.A. Hofmann, W.V. Rustrum, K.S. Suder, Electro-incorporation of microcarriers as a method for the transdermal delivery of large molecules, Bioelectrochem. Bioenerg. 38 (1995) 209–222. [11] L. Zhang, L.N. Li, Z.L. An, R.M. Hoffman, G.A. Hofmann, In vivo transdermal delivery of large molecules by pressuremediated electroincorporation and electroporation: a novel method for drug and gene delivery, Bioelectrochem. Bioenerg. 42 (1997) 283–292. [12] W.R. Ravis, Y.J. Lin, R. Murty, in: D.L. Wise (Ed.), Handbook of Pharmaceutical Controlled Release Technology, Marcel Dekker, Inc., New York, Basel, 2000, p. 821. [13] S. Chang, G.A. Hofmann, L. Zhang, L.J. Deftos, A.K. Banga, The effect of electroporation on iontophoretic transdermal delivery of calcium regulating hormones, J. Controlled Release 66 (2000) 127–133.
Electrically-assisted transdermal delivery of buprenorphine Sagarika Bose a , William R. Ravis a , Yuh-Jing Lin a , Lei Zhang b , ¨ Gunter A. Hofmann b , Ajay K. Banga c , * a
Department of Pharmacal Sciences, School of Pharmacy, Auburn University, Auburn, AL 36849 -5503, USA b Genetronics, Inc., 11199 Sorrento Valley Road, San Diego, CA 92121 -1334, USA c Department of Pharmaceutical Sciences, School of Pharmacy, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341 -4155, USA Received 24 July 2000; accepted 20 January 2001
Abstract The objective of this study was to explore the electrically assisted transdermal delivery of buprenorphine. Oral delivery of buprenorphine, a synthetic opiate analgesic, is less efficient due to low absorption and large first-pass metabolism. While transdermal delivery of buprenorphine is expected to avoid the first-pass effect and thereby be more bioavailable, use of electrical enhancement techniques (iontophoresis and / or electroporation) could provide better programmability. Another use of buprenorphine is for opiate addiction therapy. However, a patch type device is subject to potential abuse as it could be removed by the addict. This abuse can be prevented if drug particles are embedded in the skin. The feasibility of doing so was investigated by electro-incorporation. Buprenorphine HCl (1 mg / ml) in citrate buffer (pH 4.0) was delivered in vitro across human epidermis via iontophoresis using a current density of 0.5 mA / cm 2 and silver–silver chloride electrodes. Electroporation pulses were also applied in some experiments. For electro-incorporation, drug microspheres or particles were driven into full thickness human skin by electroporation. It was observed that the passive transdermal flux of buprenorphine HCl was significantly enhanced by iontophoresis under anodic polarity. The effectiveness of electro-incorporation seemed inconclusive, with pressure also playing a potential role. Delivery was observed with electro-incorporation, but the results were statistically not different from the corresponding controls. 2001 Elsevier Science B.V. All rights reserved. Keywords: Iontophoresis; Electroporation; Buprenorphine; Skin transport
1. Introduction Buprenorphine is a narcotic analgesic which is approximately 30–50 times more potent than morphine. It is commercially available (Buprenex ; Reckitt and Colman, VA) for relief of moderate to *Corresponding author. Tel.: 11-678-547-6243; fax: 11-678547-6423. E-mail address: banga [email protected] (A.K. Banga). ]
severe pain. It is administered parenterally by intramuscular or slow intravenous injection at a typical dose of 0.3–0.6 mg every 6–8 h. The patient may have to take the injection 3–4 times a day, depending on the severity of the pain. Thus, controlled release parenterals or alternate delivery systems are desirable. Oral delivery is impractical as buprenorphine undergoes an extensive first pass metabolism. Buccal or oral mucosal delivery of buprenorphine has been investigated [1,2] and a sublingual system
0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00298-X
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S. Bose et al. / Journal of Controlled Release 73 (2001) 197 – 203
has been commercialized in Europe. Nearly 75% of buprenorphine was shown to release from a bioadhesive Carbopol -based buccal patch following a 24 h incubation in phosphate buffer [1]. Transdermal delivery offers a viable alternative [3–6]. A prodrug strategy has been used with some success where the drug is metabolized to the parent compound while in the skin [5]. However, it has been shown that transdermal delivery of buprenorphine hydrochloride by itself can also result in adequate plasma levels for sustained analgesic effect. There was a delay in onset which could be reduced by using an ethanolic solution [6]. Electrically-modulated delivery [7] by iontophoresis and / or electroporation will provide a further advantage of programming the dose in proportion to the level of analgesia desired. Buprenorphine can also be used for opiate addiction therapy [8] as it has lower abuse potential and milder withdrawal symptoms compared to methadone. For this use, a patch type device may not be desirable as it is subject to potential abuse since it could be easily removed by the addict. If buprenorphine drug particles can be successfully incorporated into the skin, then the drug may release from the formulation for an extended period of time, and the advantage of this approach would be that the formulation cannot be easily removed by the patient / addict. Buprenorphine is a very lipophilic drug. Its hydrochloride salt still remains lipophilic despite the small hydrophilic addition (HCl) which is not able to shield the bulky hydrophobic moiety. However, the lipophilicity is reduced for the HCl salt which is able to better permeate the skin than the free base as it can partition out of the epidermis into the dermis more easily due to its moderate lipophilicity [3]. There is one report in the literature on the iontophoretic delivery of buprenorphine to weanling Yorkshire swine [9]. Though therapeutic levels were achieved, the flux was very low and a strong skin depot was observed. This could be because the drug is very lipophilic. The use of electroporation alone or in combination with iontophoresis to deliver buprenorphine has not been investigated. Electroporation directly enhances the permeability of the stratum corneum, the outermost resistive layer of the skin, by creating new pathways in addition to existing ones.
2. Materials and methods
2.1. Materials Buprenorphine HCl (MW 504.11) and 3 H labeled buprenorphine was obtained from RBI (Natick, MA) and Sigma (St. Louis, MO), respectively. Sodium chloride, sodium phosphate monobasic and dibasic citric acids were obtained from Fisher Scientific (Pittsburgh, PA). Scintillation cocktail (ULTIMAGOLDE) and Solvable tissue and gel solubilizer was purchased from Packard (Meriden, CT). Silversilver chloride electrodes were obtained from In Vivo Metric (Healdsburg, CA). Silver wires were obtained from Aldrich Chemical Co. (Milwaukee, WI). Human cadaver skin was obtained from the Cooperative Human Tissue Network (Birmingham, AL) and was frozen within 12 h of death and supplied as full thickness skin. Pig skin was obtained from Auburn University, College of Veterinary Medicine (Auburn, AL). Neonatal pigs were used (Genus species: Sus scrofa) to procure skin as these were readily available. All the skins were frozen at 2808C and thawed just before use.
2.2. Preparation of skin For full-thickness skin (human and pig), the abdominal skin was thawed before use by immersing it in distilled water at room temperature. The underlying subcutaneous fat was gently scraped off till the skin was about 1–1.5 mm thick. Then it was cut into appropriately sized pieces and mounted on Franz transdermal diffusion cells. For removal of epidermis from full-thickness human skin, the skin was heated in water at 608C for 45 s and then rubbed gently by two spatulas. The epidermal membrane was teased off from the underlying dermis by forceps. Then, it was spread over a piece of parafilm so that it becomes easy to cut into appropriate pieces.
2.3. Transdermal studies Franz (vertical) diffusion cells were used for in vitro permeation studies. The donor half was exposed to room temperature (258C), while the receptor half was maintained at 378C. The skin was
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thawed and mounted on the diffusion cells. The receptor phase was filled with 5.0 ml of pH 6.0 phosphate buffer (as buprenorphine is soluble at this pH). The donor phase (0.5 ml) was made up of cold buprenorphine (1 mg / ml) spiked with tritiated buprenorphine in pH 4.0 citrate buffer. Samples (0.5 ml) were taken from the receptor phase and analyzed by LSC. Experiments were performed in triplicate and all data are plotted as mean6standard deviation. For iontophoresis, silver–silver chloride electrodes were used because they do not cause electrolysis of water, which may result in pH shifts. Silver wire was used as the anode and silver–silver chloride was used as the cathode. The anode was placed in the donor solution and the cathode was placed in the receptor unless otherwise specified. A current of 0.5 mA / cm 2 was applied for 4 h and sampling was continued for 24 h. For Electroporation, platinum wires were used as electrodes (both cathode and anode), and the exponential pulse generator was a BTX Electro Cell Manipulator Model 600 (Genetronics Inc., San Diego, CA). High voltage (.100 V) pulses (10 or 20 ms) were applied to the skin. The pulse rate or the number of pulses per minute was manually controlled as the protocols of the experiments were changed. Anode was placed in donor chamber and cathode was placed in receptor. In some experiments, the combined use of iontophoresis and electroporation was explored.
2.4. Electro-incorporation studies Electro-incorporation [10,11] studies were performed using either microspheres or drug particles (solid state) placed on full thickness human skin. Buprenorphine microspheres were prepared by the method of Ravis et al. [12] by solvent evaporation methods, using PLGA (65:35) as the biodegradable polymer. Briefly, Buprenorphine HCl was dissolved in methylene chloride with PLGA polymer. The solution was added to purified water and following mixing by vortexing, the dispersion was added to a solution of poly(vinyl alcohol) and mixed for 3 h. After centrifuging, the microspheres were washed with water. Buprenorphine was labeled with 3 Hbuprenorphine in the microspheres. For preparation
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of drug particles, buprenorphine was dissolved in ethanol and tritiated buprenorphine (in ethanolic solution) was added to this. The ethanol was then evaporated under nitrogen to get an intimate mixture of cold and hot buprenorphine. This was suspended in pH 7.4 phosphate buffer (1 mg / 6 ml of suspension), as buprenorphine is practically insoluble at this pH. The microsphere or drug particles’ suspension (6 ml) was placed on skin and covered with a meander electrode. A meander electrode consists of an interweaving array of metal fingers coated on a thin plastic film which can be placed on the skin. Electroporation pulses were applied via these metal fingers on the meander electrode. This creates an electric field which breaks down the stratum corneum by a yet unknown mechanism. The electrode is in direct contact with the skin here and low voltage is given so that it does not damage the skin. A slight pressure was maintained on the electrode during pulsing by clamping the whole set-up on the upper half of a Franz cell. The drug suspension under the meander electrode substitutes for the donor solution and the upper half of the Franz cell is being used merely to hold the meander electrode in place. The skin was covered with parafilm and a metal plate before clamping to maintain some pressure on it. Meander electrode was connected to the electroporation equipment and pulses were given. After pulsing, the skin was washed several times with pH 4.0 citrate buffer to remove any drug adhering to the surface as buprenorphine is more soluble at this pH. The skin was then stripped five times with tape. The remaining skin was digested with Solvable , a tissue and gel solubilizer at 708C until the skin had completely dissolved. The residual radioactivity in the skin and strips was then assayed separately by liquid scintillation counter (LSC).
2.5. Skin impedance studies The electrical properties of the skin in the passive state were measured using a function generator (V0 5 1V; repetition at f51 KHz; sine wave). The resistance of the chamber without the skin, R Bulk , was also measured in a similar fashion. The skin was pulsed using the BTX pulser. To measure the applied
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voltage (V0 ), a digital oscilloscope was used and the trace stored in channel 1. The voltage developed across the 15 V resistor in series with the chamber was measured at channel 2 (Vs ). This would give the total current through the chamber. The effective transdermal voltage (VSkin ) was calculated and was much lower than the applied voltage across electrodes (Velectrode ), a typical VSkin being about 50 V for an applied Velectrode of 500 V. The voltages are given as Velectrode in this paper.
3. Results and discussion
3.1. Transdermal transport studies The effect of formulation pH on iontophoretic transport of buprenorphine was investigated. With the pH of donor solution at 4.0, the average cumulative amount in the receptor was 15.8961.94 mg / cm 2 at 24 h while it was only 5.161.8 mg / cm 2 when the donor pH was 5.0. This may relate to the change in the charge of the skin (and hence electroosmotic flow) at different pH or to the higher solubility of buprenorphine at pH 4.0. Fig. 1 shows the average cumulative amount present in the receptor during anodic (anode in donor) and cathodic (cathode in donor) delivery over 24 h following a 4-h current application. The amount delivered under anode (15.8961.94 mg / cm 2 ) was much higher than that delivered under cathode (1.9060.06 mg / cm 2 ). The results are statistically significant (P,0.05). Passive delivery (no current) resulted in very low permeation, similar to cathodic delivery. Buprenorphine has a positive charge at pH 4.0 and this study establishes that it should be delivered under anode. The permeation of buprenorphine seems to continue even after stopping the current. This could be due to a skin depot (reservoir) which is slowly released into the receptor. Alternatively, the skin may have been compromised by iontophoresis so that the drug is diffusing from the donor to the receptor. The latter is not expected at the low current density used. However, to investigate which one of these is most likely explanation, a desorption study was done in which drug solution in donor is replaced with buffer after 4 h of iontophoresis and then sampling was continued to analyze the drug being released from the depot
Fig. 1. Comparison between anodic, cathodic, and passive transport of buprenorphine after 4 h of iontophoresis (0.5 mA / cm 2 ) followed by passive delivery upto 24 h through full thickness pig skin (n53).
(desorption). Desorption was carried out under passive (no current after 4 h) or iontophoresis (anodic iontophoresis reapplied after replacing drug solution with plain buffer) conditions (Fig. 2). The data shows that buprenorphine was released into receptor even after donor solution was replaced with buffer, suggesting the presence of a skin depot. The difference between passive and iontophoretic desorption was not statistically significant (P,0.05), suggesting that the skin reservoir of lipophilic buprenorphine cannot be pushed out by the iontophoretic current used in these experiments. Fig. 3 shows the difference between iontophoresis, iontophoresis coupled with electroporation, and electroporation alone using full thickness pig skin. All the results are statistically significant (P,0.05). The electroporation protocol was 20 pulses (10 ms each) of 500 V (applied voltage) at time zero while iontophoresis current was applied for 4 h. Electroporation alone was unable to enhance transport perhaps because there is no sufficient driving force (as in iontophoresis) to push the drug into the skin permeabilized by electroporation. In addition, the total amount of charge delivered by
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Fig. 2. Desorption of buprenorphine reservoir in skin under passive or iontophoretic conditions following 4 h (0.5 mA / cm 2 ) of iontophoretic delivery of buprenorphine (n53).
Fig. 3. Effect of iontophoresis for 4 h (0.5 mA / cm 2 ) and electroporation (applied voltage 500 V, 10 ms, 20 pulses) followed by passive delivery for 24 h on buprenorphine delivery through full thickness pig skin (n53).
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electroporation is significantly less than that delivered by iontophoresis. A combination of the two modalities resulted in delivery which was over six times higher than that achieved by electroporation alone and about twice of that achieved by iontophoresis alone. In another study, a comparison between only passive transport and passive transport after electroporation was investigated using pig skin. The average cumulative amount in the receptor after 24 h for passive transport was 1.7860.69 mg / cm 2 while when 20 pulses (each of applied voltage 500 V, 10 ms) were given at time zero, the average cumulative amount in the receptor was 1.9660.41 mg / cm 2 after 24 h. Thus, electroporation only does not facilitate the transport to a significant extent. Iontophoretic delivery of buprenorphine through human epidermis is shown in Fig. 4. As can be seen, the depot effect seems less when epidermis rather than full thickness skin is used. In practical terms, this result is more meaningful since the blood circulation lies just under the epidermis and will drain most of the drug quickly, preventing the formation of a skin depot. Delivery through epidermis was also higher than
Fig. 4. Effect of iontophoresis for 4 h (0.5 mA / cm 2 ) and electroporation (applied voltage 500 V, 10 ms, 20 pulses) followed by passive delivery for 24 h on buprenorphine delivery through human epidermis (n53).
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that through full thickness skin. By extrapolating the data to typical patch sizes available on the market (about 5–30 cm 2 ), it can be seen that therapeutic doses can be easily delivered. In the case of human epidermis, electroporation failed to enhance iontophoretic delivery as used in this study. However, this could be drug and protocol specific as for calcitonin [13], electroporation did enhance iontophoretic transport across human epidermis. The impedance of the skin was measured for one protocol (applied voltage 500 V, 10 ms, 4 ppm for 30 min). The initial impedance of the skin was 6.7 kV and it fell to about 30 V during the pulses. Following electroporation, it recovered to about 1.6 kV in 24 h. The effect of number of pulses (applied voltage 500 V, 10 ms) applied was also investigated, and no difference was observed when 50 or 100 pulses were applied prior to 4 h of iontophoresis (data not shown).
3.2. Electro-incorporation studies Buprenorphine microspheres were pulsed on human full thickness skin by meander electrodes using six pulses (applied voltage 120 V, 10 ms each) at time zero. Control experiment was treated with the formulation and electrodes were placed the exact same way but no pulses were applied. The microspheres were 5–20 microns in diameter and the final product contained 7.94% drug. The electroporation applied voltage (120 V) was lower than what was used in solution studies as these electrodes are in direct contact with the skin. The results (Fig. 5) show that there was no significant difference between pulse and control experiment. In a study with a suspension of pure drug, the same amount of solid placed under meander electrode and pulsed with the same protocol as for microsphere formulation. The use of pure drug suspension resulted in significantly higher delivery. Furthermore, the experimental group had significantly higher drug than the control. A more intense protocol (20 pulses; applied voltage 120 V, 20 ms each) resulted in somewhat higher delivery but now the difference between control and experimental groups was not statistically significant (P,0.05) (Fig. 6). Considering that no pulses were given, the control values were very high in these electro-incorporation experiments. This was initially suspected due to inadequate washing protocol. How-
Fig. 5. Delivery of buprenorphine microspheres through human full thickness skin by electroincorporation (applied voltage 120 V, 10 ms, 6 pulses) (n53).
ever, careful and extensive washing did not help much to resolve the problem. Skin stripping was performed and it was observed that the control skin had significant amount of drug even after stripping. It is possible that particles sticking to the skin may
Fig. 6. Delivery of buprenorphine solid drug suspension through human full thickness skin by electroincorporation (applied voltage 120 V, 6 pulses) (n53).
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study had relatively small effect on drug delivery. The feasibility of delivery of buprenorphine particles (microspheres or pure drug suspension) in skin by electroporation could not be established in this study with the electrical protocols used. References
Fig. 7. Amount of drug transported in 48 h following electroincorporation (applied voltage 120 V, 10 ms, 12 pulses) of buprenorphine microspheres in full thickness human skin (n53).
be driven into the skin by pressure alone. Such pressure-mediated delivery was not investigated in these experiments. Alternatively, some drug could also release from the surface of microspheres and then enter the skin. In one study, an indirect measurement of electro-incorporation was attempted by leaving the skin on Franz diffusion cells for 48 h after pulsing (12 pulses of applied voltage 120 V, 10 ms), with skin being exposed to the receptor solution. Samples were taken from the receptor and analyzed over this time period. The results (Fig. 7) show that the permeation between control and pulsed groups was statistically insignificant (P,0.05).
4. Conclusion The passive transdermal flux of buprenorphine HCl is significantly enhanced by iontophoresis. Anodic delivery was higher than cathodic delivery for a pH 4.0 formulation. A depot (reservoir) in the skin was observed following iontophoretic drug delivery. The electroporation protocols used in this
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