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I r~ ii FJ-SEVMR journal of controlled release Journal of Controlled Release 49 (1997) 177-184 In vitro skin permeation of nicotine from proliposomes Bo-Young Hwang, Byung-Hwa Jung, Suk-Jae Chung, Min-Hwa Lee, Chang-Koo Shim* Depaia„uu of Pharmaeerrticr, College of Pharmacy, Seoul Natiortal Univeraity, Seaul 151-742 South Korea Received 30 October 1996; received in revised form 30 March 1997; accepted 14 April 1997 Abshad The feasibility of proliposomes as a sustained transdermal dosage form was examined. Proliposomes containing varying amount of nicotine were prepared by a standard method using sorbitol and lecithin. The porous structure of sorbitol in the proliposomes was maintained, indicating that the majority of lecithin and nicotine is deposited within their porous matrix of the sorbitol particles. As a conseqttence, the flow properties of the proliposome particles was comparable to that of original sorbitoi particles. Microscopic observation revealed that proliposomes are converted to liposomes almost completely within minutes following contact with water. It indicates that proliposomes may form liposomes by the sweat when they are applied on the skin under occlusive conditions in vivo. The size distribution of the reconstituted liposotaes and nicotinarelease to pH 7.4 pho5phaie buffer from them were not significantly affected by the content of nicotine. The release pattern was apparently ~ ideadcal to the Exodus' patch, a commercially available transdermal nicotine formulation. We also studied in vitro permeation of nicotine across rat skin from proliposomes in a modified Keshary-Chien diffusion cell where the experimental J f 7 set up simulates in vivo application of the proliposomes under an occlusive condition. The nicotine flux from proliposomes was initially retarded compared with that of nicotine powder. The flux from proliposomes appeared to remain constant thtvughqut the experimental period compared with that of nicotine powder, indicating that nicotine may be delivered across the skin in a sustained manner at a constant rate from proliposomes. These results, therefore, indicate that sustained traasdermal delivery of nicotine is feasible using proliposomal formulations if the formulations are topically applied under occlusive conditions. ® 1997 Elsevier Science B.V. Keywords: Nicotine; Sustained release; Transdermal delivery; Liposome; Proliposome 1. Introduction Proliposomes are free-flowing particles which immediately form a liposomal dispersion upon hy- dration [1,2]. They are composed of drug(s), phos- pholipid(s) and a water-soluble porous powder. Proliposomes can be stored sterilized in a dried state [3,41. Moreover, by controlling the size of the porous `Cortesponding author. Tel. +82 2 880 7873; fax: + 82 2 885 8429. powder in proliposomes, relatively narrow range of reconstituted liposame size can be obtained [5]. Because of these properties, proliposomes appear to be an elegant alternative to liposomes in design and fabrication of liposomal dosage forms. When proliposomes are applied to mucosal mem- branes, they are expected to form liposomes upon hydration by mucosal fluids. The resulting liposomes may act as a sustained release dosage form of the loaded drugs. As a matter of fact, blood concen- tration of propranolol could be sustained substantial- 0168-3659/97/517.00 © 1997 Elsevier Science B.V. All rights reserved P11 S0168-3659(97)00073-4 nus wUe is to, ind,wdual uu ody and may na be fimhu repnduoed « samd d«vomaly wd,aa wncm pmraoon som the eopyr,ghe notder U(w+dionzed repioeucem mry rcsuh in Cmu+aat +d otha penaliea (c) ELSEV[ER SC[ENCE B V

® 178 B. HwaR g er al. / Journal of CorurolL-d Release 49 (1997) 177-184 ly by intranasal administration of a proliposomal powder (6). Stimulated by these findings, we wished to extend the application of proliposomes to systemic delivery of drugs across the skin. This would be possible if proliposomes form liposomes upon hydra- tion by the sweat on the skin following topical application under occlusive conditions. The purpose of this study is to examine the feasibility of such proliposomes as a transdermal drug delivery system. Nicotine was selected as a model drug to be delivered in a sustained fashion since it is absorbed fairly well and eliminated rapidly when applied topically [7] and transdetznal nicotine therapy is widely used to aid smoking cessation. If nicotine can be delivered transdermally from proliposomes in the form of patches, for example, sustained absorption of nicotine would be achieved without using sophisti- cated devices and/or artificial membranes since proliposomes themselves may act as a release rate controlling dosage form of nicotine. In this respect, proliposomes composed of nicotine, sorbitol (core material) and lecithin (liposome-forming lipid) were prepared and skin permeation of entrapped nicotine from the proliposomes was evaluated in vitro. Some pharmaceutical characteristics of the proliposomes such as surface morphology, in vitro drug release, conversion to liposomes upon hydration, size dis- tribution and drug entrapment efficiency of reconsti- tuted liposomes were also examined. We report that sustained delivery of nicotine across the skin could be achieved through topical application of nicotine- loaded proliposomes under occlusive conditions in vitro. 2. Materials and methods 2.1. Materials Nicotine was purchased from Fluka AG Co. (Switzerland). Egg lecithin [type X-E from dried egg yolk, the phosphatidylcholine content of approxi- mately 60% (w/w)] was purchased from Sigma Chemical Co. (St Louis, MO. USA). Sorbitol was purchased from Junsei Chemical Co. (Tokyo, Japan) and the fraction with a particle size of 105-350 µm was collected by sieving. Gentamicin and normal saline were purchased from Yuhan Pharmaceutical Co. (Seoul, Korea) and Choong Wae Pharmaceutical Industry Co. (Seoul, Korea). Silicon oil was pur- chased from Dow Corning Co. (Midland, MI). Reagents and organic solvents such as ammonium molybdate, sodium acetate, glacial acetic acid, potas- sium phosphate monobasic, triethanolamine, ether and methanol were all reagent grade or better. 22. Preparation of nicotine-loaded proliposomes A modified rotary evaporation unit (Eyela, Tokyo R91rnlc; ai Co. Ltd., Tokyo, Japan) was used for the preparation. A polypropylene tube connected to a two-way cock was inserted into the evaporator via solvent inlet tube. Sorbitol (10 g, particle size 105- 350 Es.m) was placed in a 100 ml round-bottomed flask which was held at 70-80°C in a thetmo- statically controlled water bath and the flask was rotated at 80-90 rpm for 30 min under vacuum. After the sorbitol in the flask dried, the,temperature of the water bath was lowered to 20-30°C. Egg lecithin (1 g) and nicotine (83 mg or 162 mg) were dissolved in 10 ml of chloroform. A 0.5 ml aliquot of each chloroform solution was introduced slowly into the flask via the solvent inlet tube. The solution was allowed to be absorbed into the microporous sorbitol. When the sorbitol had good flowability after drying, a second aliquot (0.5 ml) of the solution was introduced. This process was repeated until the solution (10 ml) was used up. After drying, the vacuum was released and the drying process was completed by connecting the flask containing proliposomes to a lyophilizer overnight. The fraction of proliposomes 105-350 µm in diameter was collected using appropriate sieves. Subsequently, the proliposomes were transferred into a glass bottle, flushed with nitrogen, sealed and stored in the freezer until characterization. The proliposome par- ticles were almost free-flowing. 23. Scanning (SEM) and transmission electron microscopy (TEM) Surface morphology of proliposomes was ex- arnined by a scanning electron microscopy (SEM). Proliposomes and sorbitol particles were coated with platinum/palladium in a sputter coater (Jeol Fine Coat Ion Sputter, JFC-1100, Japan) and their surface t

0 B. Hwang et al. I Journal of Controlled Release 49 (1997) 177-184 279 , -i 0 I a i 1 I morphologies were photographed with Jeol scanning elxu+on microscope (JSM-35, Japan) at 1000X. Formation of liposomes from proliposomes upon hydnation was examined by optical microscopy and transmission electron microscopy (TEM). A drop of distilled water was added to the proliposome graauies on a slide glass without a cover slip, and the process of liposome formation was observed through an optical microscope at 1000X. Samples for TEM were prepared by adding I ml of phosphate buffer (pH 7.4) to 100 mg proliposomes and vortexing the mixture for 30 sec. A drop of the sample was placed onto a carbon-coated copper grid to leave a thin film on the grid. Before the film dried on the grid, the film was negatively stained with 2% (w/w) am- monium molybdate in 2% (w/v) ammonium acetate buffer (pH 6.8). A drop of the staining solution was added onto the film, and the excess of the solution was removed with a filter paper. The grid was allowed to be air-dried thoroughly and samples were viewed on a transmission electron microscope (Jeol- 200 CX, Jeol, Japan) and photographed under 200 KV at 20 000X. 2.4. Particle size determination of reconstituted lipasomes Ptn 'hposomes were mixed with distilled water to ~ malte 5% (w/v) suspension with manual agitation for 1 min twice with a 15 min interval. The size disaribution of the resulting liposomes was character- ized using a laser particle analyzer (LPA-3 100, Phtal ~ Ostuka Electric Co. Japan). 2..5. Nicotine content in proliposomes and lfposomes after hydration I Nicotine content in proliposomes was assayed by an HPLC method (for detail, see section 2.8). Proliposomes (100 mg) were dissolved in the mix- ture of phosphate buffer (pH 7.4, 1 ml) and methanol (9 ml) by vortexing, and 25 µl of the resultant solution was injected onto the HPLC column. An arlritrary index was introduced to express the etm'apment efficiency (EE) of nicotine in liposomes following reconstitution from proliposomes. The entrapment efficiency was determined as follows: proliposomes were mixed with distilled water to make a 0.5% (w/v) suspension and the suspension was shaken manually for i min twice with a 15 min interval for complete hydration. A 100 µ1 aliquot of the resultant dispersion was mixed with 2.0 ml methanol, and 20 µ.1 of the mixed solution was injected onto the HPLC column to determine nico- tine in the dispersion. The remaining dispersion was centrifuged for 60 mi.n at 13 000Xg and 20 µ1 supernatant was injected onto the HPLC column. The EE of nicotine was calculated as follows: EE(%) = 100 X (A - B)/A (1) where A and B represent the nicotine concentration in the dispersion before centrifugation and that in the supernatant after centrifugation, respectively. During the above process, however, release of the entrapped nicotine from the liposomes may occur. Therefore, the EE in this calculation may reflect the retained nicotine in the liposomes after the treatment rather than entrapped nicotine itself. 2.6. Release of nicotine across the semipermeable mernbrant Release of nicotine across a semipermeable mem- brane from the nicotine-containing proliposomes was determined using a USP dissolution apparatus (DST- 200, Fine ittstrument, Seoul, Korea) equipped with a rotating paddle. 1 g of nicotine-loaded proliposomes was put into a clean dialysis bag (Spectra/Por 2 membrane, m.w, cut off of 12 000-14 000; Spectra Medical Ind., Los Angeles, CA, USA). The bag was secured with two clamps at each end to yield a rectangular shape of 3.9 X 5.0 cm and placed into the release apparatus containing 500 ml phosphate buffer (pH 7.4). The buffer was kept at 37-* 1°C and stirred with the paddle at 100 rpm. At 15, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after placing of the bag in the release medium, 1 ml aliquots of the medium were sampled with the replacement of the fresh medium. The samples were stored in a freezer prior to analysis of nicotine. For comparison, nicotine powder (1 g), instead of proliposomes, was placed in the dialysis bag and the release of nicotine across the bag was tested in the same manner. Release of nicotine from a commercial nicotine patch, Exodus® (Elan Pharm. Co., Ireland, nicotine 30 mg, transpassing area: 7 cm2), was also N m 0

® E 180 B. Hwang et al. / Journal of Corstrolled Release 49 (1997) 177-184 determined by the paddle over disk method of USP for transdermal patches [8]. 2.7, In vitro skin permeation study 27.1. Preparation of rat skin Abdominal skin of Wistar male rats was used in the study. Rats (250-280 g) were anesthesized slightly by ether and hairs were removed from the abdominal skin with the aid of an electric animal clipper (Daito Electric Mfg. Co., Japan) and shaver. Care was taken not to damage the skin surface. The rats were sacrificed by air injection via the femoral vein and the abdominal skin of the rat was separated. The skin was stored at -20°C until the permeation study. Before the permeation study, the skin was hydrated in normal saline (contained 200 ppm gen- tamicin) at 4°C and the adipose tissue layer of the skin was removed by rubbing with a cotton swab. 27.2. Permeation of nicotine from proliposomes under occlusive condition To simulate the in vivo situation where prolipo- somes are applied onto the surface of skin under occlusive condidons, nicotine powder (2.0 mg) or proliposome particles equivalent to 2.0 mg of nico- tine were evenly spread on the skin surface of circular area of 2.16 cm2 and covered tightly with an occlusive film (Transpaseal®, Porcupine Canvas, Ontario, Canada). The circular rim of the dosing site was sealed with silicon oil in order to prevent leakage of the proliposome particles during the permeation study. Care was taken so that the silicon oil was not spread into the dosing area. Then the proliposome-loaded skin was mounted carefully on the Keshary-Chien diffusion cells (K.C. Scientific Co., Korea). The stratum comeum side and dermal side of the skin were located in order to face the donor compartment and receptor compartment, re- spectively. The effective transpassing area of the diffusion cell was 2.16 cm2. The receptor compart- ment was filled with 10 ml of phosphate buffer (pH 7.4), and the buffer was stirred by a magnetic stirrer rotating at 600 rpm and kept at 37-!-1°C. No buffer was added into the donor compartment and, thus, the stratum corneum side of the skin was exposed to atmosphere. At 1, 2, 4, 6 and 8 h after the experi- ment start, 100 µ1 aliquot was sampled from the receptor compartment with the fresh buffer replace- ment The samples were stored in a free= prior to analysis of nicotine. 28. HPLC analysis of nicotine The concentration of nicotine in the liposomal dispersion and release medium was determined by injecting 25 µl of each solution onto a HPLC column according to the HPLC method described in [9]. The HPLC system (Schimadzu Co., Japan) consisted of LC-9A solvent delivery pump, SCL-6B system con- troller, SPD-6A UV spectropbotometric detector, C- R6A integrator and a guard column (Shim-Pack G-ODS, 1 cm X 4.0 mm i.d., 5 µm) connected to an analytical column (Shim-pak CLS-ODS (M), 25 cm X 4.6 mm i.d., 5 µ.m). The mobile phase was a mixture of 0.05 M sodium acetate and methanol (85:15), containing 0.3% triethanolamine, and pH was adjusted to 4.2 using glacial acetic acid. The flow-rate was 1.5 ml/atin and the effluent was monitored at 254 nm. Nicotine peaks were clearly separated with a retention time of 4.4 min after sample injection. Linear correlation (r=0.99996) was obtained between the nicotine concentrations (0.016-0.16 ng/ml) in the standard solutions and their peak heights. Peak height measurement was used for quantification of nicotine. 3. Results and discussion 3.1. Characteristics of nicotine-containing proliposomes Nicotine contents in the proliposomes were 1.37 and 0.693'0 (w/w) for the proliposomes of different lecithin/sorbitol/nicotine ratio (1 / 10/0.162 and 1/ 10/0.083, respectively). This implies that more than 90% (94.4 and 92.1 %, respectively) of added nico- tine was recovered in the proliposomes. The rest of nicotine not recovered might be adsorbed on to the surface of the flask where the proliposomes were prepared. Surface morphology of proliposomes of 105-350 µ.m fraction was compared with that of the untreated sorbitol of the same particle size by scanning elec- tron microscopy (SEM, Fig. 1). The porous suvcture i r

B. Hwang et al. / Journal of Controlled Release 49 (1997) 177-184 181 F'iX. 1. Scanning electron micrograph (1000x magfficaaoa) of aocbitol (3eft) and nicotine loaded peoliposome particle (right) (lecithin/ satbimi/nicaine=1 / 10/0.162). I I I I ~ j 7 7 of socbitol was partly maintained in the proliposomes which appears to explain the flowability of the proliposomes which is comparable to that of sorbitol particles. This observation is consistent with those of Payne et al [1] and Ahn et al [5]. 3.2 Reconstitution of tiposomes from proliposomes upon hydration Observation under an optical microscope revealed that proliposome particles are progressively, but rapidly (i.e. in less than 30 sec), converted to form a aemi-tlansparent mixture in water. This can be explained if reconstitution of liposomes from the proliposomes is assutned. Systems other than lipo- somes are hardly expected to solubilize nicotine and phosphatidyicholine to yield a fairly transparent mizture. However, liposomes themselves could not be observed under the optical microscope. It appears to indicate that a proliposome particle (105-350 p.m) is divided, upon hydration, into much finer particles (probably lipasomes) which cannot be observed under the microscopy. Liposome formation was supported by the size of the reconstituted particles in the mixture which was consistent with the size of liposomes. The mean diameter of the particles in- creased from 104±-18.1 to 121-!-27.3 nm as the ratio of lecithin/sorbitol/drug in the proliposomes changed from 1/ 10/0.162 to 1/ 10/0.083. It should be noted that proliposomes used in this analysis were of identical particle size (105-350 µm) irrespective of their compositions. Therefore, the above results indicate that the particle size of the reconstituted liposomes may increase as the lecithin content in the proliposomes increases. Liposome formation could be confirmed again by TEM. A TEM image of the liposomes reconstituted from the proliposomes is shown in Fig. 2. Particles having approximate diameter of 100 nm are ob- served. Therefore, we conclude that proliposomes are converted to liposomes in the presence of water. Fig. 2. Transmission electron micrograph ( x20 000 magnifica- tion) of liposomes reconstituted from proiiposomes having a composition of Iccithin/sorbitol/nicotine=1 / 10/0.162.

® ® 9 182 B. Hwang et al. / Journal of Controlled Release 49 (1997) 177-184 These results are consistent with our previous ob- servations for the proliposomes containing proprano- lol hydrochloride [5] and Sudan IV [10J. 3.3. Entrapment efficiency (EE) of nicotine in reconstituted liposomes The EE of nicotine in liposomes varied from 45.1 (±3.8) to 57.9 (±3.4) % as the composition (lecithin/sorbitol/nicotine) of the proliposomes changed from 1/10/0.162 and 1/10/0.083, respec- tively. It appears to increase as the lecithin content in the proliposomes increases, which is consistent with the hypothesis that nicotine is distributed in the lipid (lecithin) layer of the liposomes. It should be noted that EE may vary depending on the volume of water added for the determination since the release of nicotine during the determination may affect the EE. In this study, the EE was measured for proliposomes using 0.5% (w/v) suspension of proliposomes in water. If smaller volume of water was allowed for the determination, larger EE should have been obtained. When proliposomes are applied on the surface of skin as a transdermal delivery system, they may come into contact with very small volume of water (i.e. sweat). Thus, the EE in vivo is expected to be much larger than that obtained in the present condition. 3.4. Release of nicotine across the semipermeable membrane Fig. 3 shows the percentage of nicotine released across the semipermeable membrane from the nico- tine powder and nicotine-loaded proliposomes into a phosphate buffer (pH 7.4) as a function of time. Release of nicotine from the powder was rapid and reached approximately 90% of dose in I h. But that from the proliposomes was significantly retarded, indicating proliposomes can be a sustained release dosage form of nicotine. Despite of the sustained release, no significant time lag was observed for the release from the proliposomes. It may be due to rapid release of nicotine from the proliposomes into the medium which is consistent with the rather low value of EE. Although sustained release of nicotine could be achieved from the proliposomes, only 60% of dose was recovered from the release medium. It 0 0 2 4 6 Time (hr) 8 Fig. 3. Release of nicotine through a cellulose membrane (Mw. cut-off, 12 000-14 000) to the receptor fluid (phosphate buffer. pH 7.4, 10 ml, 37"_-1'C) from I g of nicodne -powder (t), proliposomes (*; kcithin/sorbitol/nicotiner1/10/0.162 O; kathin/sorbitol/nicotine=1/10/0.083) by USP paddle method (100 rpm). Release of nicotine from Exodus* patch (-A-) was also tested for comparison using USP paddle over disk method (100 rpm). Each point represents meaa:tSE of three different deusminations. can be explained by the fact that liposome particles cannot permeate across the semipermeable mem- brane. Then, part of the nicotine dose (approximately 40% of dose in this study) is likely to be retained in the liposomes probably as dissolved or entrapped. Similar release patterns were observed from the two proliposomes of different composition. Insen- sitivity of the release to the composition of the proliposomes may be explained by the following hypothesis. If nicotine is retained only in the water filled space inside the liposomes, the phospholipid layer of the liposomes may act as a diffusion (or release) barrier for nicotine, and the release of nicotine should have been affected by the com- position of the proliposomes which may result in liposomes of different bilayer thickness. Thus, our data appear to support the hypothesis that nicotine exists mainly in the phospholipid layer (surface), but not in the water space inside the reconstituted liposomes. Then the release will only be influenced by the partition of nicotine from the lipid layer to the release medium (buffer). This hypothesis is con-

i I I I I I B. Hwang et al. / Journal of Controlled Release 49 (1997) sistent with the fact that nicotine is sufficiently lipophilic to be dissolved in the phospholipid layer. The release patterns from both proliposomes were quite similar to that from Ezodus®, a commercial transdermal delivery system for nicotine. Although the similarity of the release pattern should not be overestimated since the experimental conditions were different for the proliposomes and Exodusm, it is noteworthy that release of nicotine from the Exodus® also showed a plateau at approximately 60% of the dose. . Entrapment of nicotine, probably in vehicles composing the patch, may be responsible for the incomplete release. The results in Fig. 3 appear to suggest the feasibility of proliposomes as a sustained release delivery system of nicotine as long as the low extent of release can be overcome when they are applied in vivo. Since liposome-bound drugs may penetrate into the skin along with the penetration of phosphatidyl- choline in the liposomes [11], proliposomes are expected to deliver more nicotine across the skin in vivo than observed from Fig. 3 (i.e. 609'0 of dose). Then the proliosomes may be concluded to be advantageous over the conventional patches which atz hardly expected to release all the dose in vivo. This aspect of transdermal delivery of nicotine is cuaently under investigation in our laboratory. 3.5. In vitro skin permeation study The objective of this study is to examine the feasibility of proliposomes as a transdermal dosage form. The simplest way of topical application may be a direct spread of the proliposomes on the skin followed by occlusive dressing of the dosed site. Thus, we studied the permeation of nicotine follow- ing application ofthe proliposomes on the skin under occlusive condition. However, a permeation study with Exodus® patch was not carried out since the patch could not be loaded on the Keshary-Chien diffusion cell without cutting the device, which may affect the release kinetics of nicotine substandally. Fig. 4 shows the cumulative amount of nicotine transferred from nicotine powder and proliposomes to the receptor compartment (pH 7.4 phosphate buffer) of the Keshary-Chien diffusion cell. As described in the section 2.7.2, the preparations were covered with an occlusive film and exposed to the 0 177-184 0 2 4 6 i Th,e (ttt) Fig. 4. Cumulative amount (mean=SE) of nicodne peaeaated to the receptor fluid (phosphate buffer, pH 7.4, 10 ml. 37t1'C) actoss the rat skin (2.16 cm2) from nicotine powder (2 mg) or proliposomes (equivalent to 2 mg of nicotine) in the Keshary- Chien diffusion cell. Drug was loaded on the stratum corneum side of the skin, which was exposed to the aanosphere during the ex.pe~~m The receptor fluid was stirred at 600 rpm. Each point represents mean=SE of three different determinations. ICey: nicotine powder 0; proliposomes, lecithin/sorbitol/nicodne=l/ 10/0.162, •; nicotine-oontaining leaithin/sortlitol/nicotine=1/ 10/0.083, 0. atmosphere. Throughout the experimental period, the powder showed a much larger amount of penetration than the proliposomes. The initial flux of nicotine (i.e. the slope of Fig. 4) from the powder was more than twice of the proliposome preparations (i.e. 172 µg/cm2/h for powder vs 73 µg/cm2/h for the proliposomes). However, the difference in flux was apparently reduced after 4 h. For the proliposomes, the nicotine flux was kept almost constant in all sampling times except initial period (between 0 to 1 h). This result appears to suggest a feasibility of proliposomes as a sustained transdermal delivery system. As the content of nicotine in the proliposome increased, the flux increased but no significant differences were found between the two prolipo- somes of different composition, which is consistent with similar release of nicotine from the two prolipo- somes (Fig. 3). Proliposomes should be hydrated to form lipo- somes before nicotine released and permeates across the skin. Interestingly, no time lag in the permeation of nicotine was observed (Fig. 4), indicating that all

B. Hwang er at. I Journal of Controlled Release 49 (1997) 177-184 the procedures (water permeation from the receptor compartment to the skin, conversion of proliposomes to liposomes, nicotine release from the reconstituted liposomes and permeation of the dissolved nicotine) occur very rapidly. It also appears to support further the hypothesis that nicotine exists mainly in the surface layer of the reconstituted liposomes. There- fore, conversion of proliposomes to liposomes by sweat is likely to occur readily after a topical application of the proliposomes under occlusive conditions in vivo. Taken together, retarded permea- tion of nicotine from the proliposomes may be primarily attributed to retarded release from the reconstituted liposomes rather than the reconstitution step. In conclusion, sustained delivery of nicotine ac- ross the skin appears to be achievable through topical application of nicotine-loaded proliposomes under occlusive conditions. The composition of the proliposomes did not affect the permeation signifi- cantly. However, an in vivo study should be con- ducted before concluding the feasibility of the proliposomes as a sustained transdermal delivery system. Acknowledgements The authors acknowledge financial support from RCNDD of Seoul National University, Korea. References [1) N.L Payne, L Browning, C.A. Hynes, Charactttiyatioo of paoLiposomes, J. Phacm Sci. 75 (1986) 330-333. [2l N.L Payne, P. Timmis, C.V. Ambrose. M.D. Warel, F. Ridgway, Proliposomes: A novel solution to an old problem, J. Pharm. Sci. 75 (1986) 325-329. [3] O.P. Katare. S.P. Vyas, V.K. Dizit, Proliposomes of in- domettucin for oral adminisaation, J. Microencapsularion 8. (1991) 1-7. [4] KJ. Oh, Preparation of sterilized prvliposomes, MSc thesis, Seoul National University, Seoul, 1993. [5J B.N. Ahn, S.K Kim, C.K. Shim, Preparation and evaluation containing propranolol hydrochloride, J. Microeacapsulation 12 (1995) 363-375. [6j B.N. Ahn, S.K. Kim, C.K. Shim, Proliposomes as an intranasal dosage form for the sustained delivery of propran- olol. J. Connvl. Rel. 34 (1995) 203-210. [7j J.H. Jaffe, Drug addiction and drug abuse, in: A.G. Good- man, T.W. Rall, A.S. Nies, P. Taylor (Eds.), The Pharmaco- logacal Basis of Therapeutics, 8th ed., Maxwell MacMillan (international edition), 1991, pp. 522-573. [8] USP XXI1, The United States Pharmacopeial Convention, Inc„ Mack Printing Company, Easton, PA, 1990, pp. 1581- 1583. [9) B. Berner, G.C. Mazzenga, P.M. Gargiulo, R. Steffens, A oransdermal nicotine system: Feasibility srudies, J. Control. Rel. 20 (1992) 13-20. [10J D.S. Chung, C.K. Shim, M.Fi Lee, S.K Kim, Proparation and evaluation of proliposome, Yakhak Hoeji 32 (1988) 234-238. [11] W. wohlrab, U. Lachman, J. Lasch, Penetration of lecithin from hydrocortisone-containing liposomes into human sYin, Dermatol. Monatsschr. 175 (1989) 344-347.