Fluorescent Probe Studies of the Interactions of 1-Alkyl-2-pyrrolidones with Stratum Corneum Lipid Liposomes KUNIO YONETO*, S. KEVIN LI †X ,WILLIAM I. HIGUCHI † ,WIM JISKOOT ‡ , AND JAMES N. HERRON † Received October 30, 1995, from the † Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, ‡ National Institute of Public Health and Environmental Protection, Laboratory for Product and Process Development, Bilthoven, Utrecht, Netherlands, and *Sekisui Chemical Co., Ltd., Corporate Research Institute, Medical Laboratory, Mishimagun, Osaka, Japan. Final revised manuscript received February 8, 1996. Accepted for publication February 8, 1996 X . Abstract 0 Previously, the effects of a series of 1-alkyl-2-pyrrolidones (APs; C2-C8) on the lipoidal pathway of hairless mouse skin (HMS) were studied with a parallel pathway skin model. At their isoenhancement concentrations, these 1-alkyl-2-pyrrolidones induce the same transport enhancement (isoenhancement factor, E HMS ) on the lipoidal pathway of the stratum corneum for the probe permeants studied. In the present study, the fluidizing effects of APs upon the stratum corneum lipid liposome (SCLL) bilayer were investigated under these isoenhancement conditions using steady-state anisotropy and fluorescence lifetime studies with fluorescent probes 2-, 6-, and 9-(9-anthroyloxy)stearic acids, 16-(9- anthroyloxy)palmitic acid, and 1,6-diphenyl-1,3,5-hexatriene to examine a possible correlation between the fluidizing properties of APs and their enhancement effects on transdermal drug transport. Time-resolved fluorescence decay studies were also conducted to further investigate the fluidizing properties of APs and add support to the steady-state fluorescence results. Under an isoenhancement condition of E HMS ) 10, these APs fluidized the alkyl chains of the lipids at intermediate depths (C6-C9) in the SCLL bilayer (a 40-50% decrease in the rotational correlation times) but did not significantly change the fluidity in the deep hydrophobic region of the bilayer. Three rotational correlation times were deduced from the global simultaneous analysis in time-resolved fluores- cence decay measurements. The slowest of these (greater than 1000 ns) was attributed to the global motion of SCLLs and is probably related to the static component of steady-state anisotropy. The other two rotational correlation times (on the order of nanoseconds) were in the range expected for the local motion of the fluorophores and may correspond to their vibrational and rotational motions. When the concentrations of APs were increased (increasing the E HMS value), the static component (R) decreased. This suggests that APs might induce a general fluidizing effect upon the lipid bilayer (i.e., a decrease in the order of the lipid bilayer). The decrease in the longer rotational correlation time (on the order of nanoseconds) with increasing E HMS value, on the other hand, indicates a possible increase in the “cavity volume” for the hindered motions of the fluorophores (i.e., an increase in the free volume at intermediate depths in the bilayer). Introduction The intercellular lipid domain of the stratum corneum is generally believed to be the lipoidal pathway of drug molecules diffusing through the stratum corneum. 1,2 It is also believed that the principal mechanism of action of chemical permeation enhancers is to fluidize this lipid domain. 1,3,4 Although short chain n-alkanols are good stratum corneum permeation enhancers, FTIR studies have provided no evidence of their ability to grossly fluidize the alkyl chains of the stratum corneum lipids. 5 Recently, Kim et al. 6 hypothesized that it may not be appropriate to consider the hydrocarbon regions of the bilayer in general as contributing to the barrier properties of the lipid domain. These investigators suggested that specific regions such as the semipolar interface of the intercellular lipid bilayers or the ordered hydrocarbon region near the interface might be considered the rate-limiting microenvironment of the lipoidal pathway. To examine this hypothesis, Kim et al. 6 studied the effects of the n-alkanols upon the steady-state anisotropy of fluorescent probes parti- tioned in stratum corneum lipid liposomes (SCLLs) and obtained results suggesting that the n-alkanols are effective “fluidizing” agents at intermediate depths (C2-C9) in the SCLL bilayer. In a previous study, 7 the effects of 1-alkyl-2-pyrrolidones (APs: 1-ethyl-2-pyrrolidone (EP), 1-butyl-2-pyrrolidone (BP), 1-hexyl-2-pyrrolidone (HP), and 1-octyl-2-pyrrolidone (OP)) upon the transport properties of the lipoidal pathway in the stratum corneum of hairless mouse skin (HMS) were studied with a parallel pathway transport model using a group of steroidal probe permeants. At their isoenhancement concen- trations, these APs induced the same transport enhancement effect (isoenhancement factor, E HMS ) on the lipoidal pathway of the stratum corneum for the steroidal permeants studied. In a similar study, the effects of APs upon the transport properties of large polar nonelectrolytes across the SCLL bilayer were also investigated. 8 The permeability increase for the SCLL bilayer, when each of these APs was present at its isoenhancement concentration, was determined and compared to the results obtained from transport enhancement studies with hairless mouse stratum corneum. A semiquantitative correlation was observed between the transport enhancement induced by APs on the SCLL bilayer and on the lipoidal pathway of the stratum corneum. This correlation suggests that the SCLLs can be a useful model for mechanistic studies on the properties of transdermal permeation enhancers and perhaps the transport behavior of the stratum corneum layer. In the present study, we used SCLLs as a model for the lipoidal pathway of the stratum corneum and investigated the interactions of APs on the fluidity at different depths in the SCLL bilayer. Steady-state fluorescence anisotropy studies were performed using two different types of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene (DPH), which parti- tioned deep in the hydrophobic interior of the SCLL bilayer; and a family of n-(9-anthroyloxy) fatty acids (n-AF) which partitioned at graded depths in the bilayer. The fluidizing effects of APs were investigated under the isoenhancement condition of E HMS ) 10 as part of our continuing strategy to better understand the mechanisms of action of transdermal enhancers. In addition to steady-state fluorescence measure- ments, time-resolved anisotropy measurements were also conducted under isoenhancement conditions ranging from E HMS ) 5 to E HMS ) 60 to add support to the interpretations based on the steady-state fluorescence results. Materials and Methods MaterialssThe 1-alkyl-2-pyrrolidones (APs) 1-ethyl-2-pyrrolidone (EP), 1-butyl-2-pyrrolidone (BP), 1-hexyl-2-pyrrolidone (HP), and X Abstract published in Advance ACS Abstracts, March 15, 1996. © 1996, American Chemical Society and 0022-3549/96/3185-0511$12.00/0 Journal of Pharmaceutical Sciences / 511 American Pharmaceutical Association Vol. 85, No. 5, May 1996 + +