2025 Volume 73 Issue 4 Pages 291-297
In this study we have focused on three types of alcohols: ethanol (EtOH), 2-propanol (IPA), and 1-propanol (NPA), and examined the skin permeability of drugs with different physicochemical properties: ketoprofen (KPF; hydrophobic), cimetidine (CMT; slightly hydrophobic), and caffeine (CF; hydrophilic). The results revealed EtOH particularly enhanced the skin permeation of CF, while IPA enhanced skin permeation regardless of the type of drug. In contrast, NPA significantly increased the skin permeability of KPF and CMT, but had little effect on CF. The differing effects of the alcohols on skin permeation appear to be linked to the physicochemical properties of the drugs. KPF is more hydrophobic than the other drugs, suggesting that it uses the intercellular pathway in the stratum corneum for permeation. CMT has intermediate properties between hydrophilic and hydrophobic, resulting in low skin permeability and ineffective utilization of both the transepidermal and transappendageal pathways. CF mainly utilized the transappendageal pathways for skin permeation because of its smaller molecular weight and more hydrophilic as compared with the other drugs. These results suggest that the effect of different alcohols on enhancing drug skin permeation is not uniform and that the optimal alcohol for enhancing permeability may vary depending on the drug. Therefore, the selection of appropriate additives based on the physicochemical properties of the drug, such as hydrophilicity, hydrophobicity, and molecular weight, is crucial for developing effective transdermal formulation.
Recently, owing to the increase in the elderly population, the development of drugs that are easy for patients to use and have various functions has increased.1) Oral formulations like tablets face challenges such as the first-pass effect of the liver and gastrointestinal issues.2) Therefore, a drug that is absorbed from the skin, called a transdermal drug delivery, is currently being considered. This methodology escapes from first-pass effect of liver, avoids swallowing difficulties, and offers benefits like pain-free administration, controlled drug release, reduced dosing frequency, and improved QOL.3) However, it is difficult to deliver the required amount of drug to the systemic circulation through the skin under normal conditions as the stratum corneum (SC), a thin film on the outermost layer of the skin, prevents the invasion of administered substances.4)
The skin consists of epidermis, dermis, and subcutaneous tissue, with the epidermis primarily composed of keratinocytes.5) The SC, the outermost layer of the epidermis, contains corneocytes embedded in a lipid matrix, which acts as the skin’s primary permeability barrier.6,7) This matrix consists of ceramides, cholesterol, and fatty acids and forms a “brick-and-mortar structure,” where keratinocytes serve as bricks and lipids as mortar, creating a strong barrier.4,8,9) Therefore, it is difficult for drugs to pass through the skin.4,10) However, it is known that even substances with a molecular weight (MW) of several hundred thousand can be absorbed into the skin when the SC is removed.10,11) The pathways through which drugs pass through the skin are thought to be transcellular pathway, intercellular pathway, and appendageal pathway.8) Absorption from the appendageal pathway can directly transition to the dermis without passing through the SC, which has a strong barrier function. However, as the surface area of the appendages is about 0.1% of the total skin area, it is reasonable to think that it slightly contributes when passive diffusion causes skin permeation. Alternatively, the pathways through the SC include the transcellular and intercellular pathways, with the latter being particularly significant among the pathways for small molecular drugs frequently utilization in clinical.4,8,12,13)
To overcome the barrier function of the SC and enhance the skin permeation of drugs, many studies have been conducted on technologies that reversibly alter the barrier function.14–16) These methods are categorized into physical and chemical approaches. Physical methods include electroporation,17) which temporarily allows a strong current through the skin, and iontophoresis,18) which uses the electrical currents to drive ionized drugs into the body. Chemical methods include utilization of prodrug,19) which introduces a functional group that is easily distributed to the skin into a specific functional group of the drug, and a method of formulating a compound that has the effect of enhancing the skin permeation of the drug.9) In general, it has been reported that compounds such as alcohols, fatty acids, surfactants, and terpenes enhance the skin permeation of drugs.4,8) Moreover, there are reports that o-ethylmenthol, a derivative of l-menthol classified as a terpene, causes lipid swelling in the SC and disrupts the lipid structure, thereby enhancing drug distribution and diffusion.20)
To enable drugs to pass through the skin, formulations must be matched to their specific characteristics. Actually, due to their varying MWs and partition coefficients (log P), the distribution of SC differs, and the pathway they take within the SC can vary depending on the drug.21,22)
Alcohol is one of the compounds that are often used as formulation components in transdermal formulations. It is expected to have a drug-dissolving effect in the formulation as a solvent and improve the skin permeability of drugs by affecting the skin itself.23) Ethanol (EtOH), a typical short-chain alcohol, has an MW of 46.07, log P-value of −0.31, and is a primary alcohol.24) 2-Propanol (IPA) is a secondary alcohol with an MW of 60.10, log P-value of 0.05.25) It has a hydroxyl group and a hydrophobic group, an isopropyl group, thus showing amphiphilicity. EtOH and IPA are classified as the same alcohol and are widely used in transdermal formulations.
In this study, we decided to focus on hydrophobicity and MW as parameters derived from the structure of the drug with regard to the skin permeability of drugs. Representative short-chain alcohols, EtOH, IPA, and 1-propanol (NPA), a structural isomer of IPA, with an MW of 60.10, log P value of 0.25 were selected,24,25) and compared the differences in the skin permeability-enhancing effects of model drugs (ketoprofen, cimetidine, and caffeine), and investigated their detailed mechanisms of action to obtain basic information for the development of effective transdermal formulations.
The alcohols (EtOH, IPA, NPA) used in this study, hydroxypropyl cellulose (HPC, MW ≤1000000) and hydroxyethyl cellulose (HEC) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). These were reagent grade. In regard to ketoprofen (KPF), cimetidine (CMT), caffeine (CF) which were used as model drugs, KPF was purchased from Sigma-Aldrich Japan LLC (Tokyo, Japan), CMT and CF from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The chemical structures of the alcohol and model drugs are shown in Fig. 1.
The concentration of alcohols was 20 and 40%. HPC, HEC, model drugs were 1, 1, 5%, respectively. The remaining was adjusted by adding purified water to achieve the total to 100%. For hydrogels without alcohols, the composition of HPC, HEC and model drugs remained the same, while purified water was added to achieve the total to 100% in the same way.
Purified water was added to the HPC and HEC and left overnight to swell as bases. Separately, model drugs dissolved in alcohol were added to the swollen base at a stirring speed of around 500 rpm. This operation was carried out at room temperature (approximately 25 °C). The gel was stored in a sealed container in a cool, dark place prior to use. The appearance of prepared hydrogels was homogenous and uniform.
In Vitro Skin Permeation ExperimentsThe hairless mice skin (Labskin® Hos: HR-1, 7 weeks old, male) was purchased from Sankyo Lab Service Co., Ltd. (Tokyo, Japan), and was placed in a Franz-type diffusion cell (effective diffusion area; 2.01 cm2) with the SC side facing the donor cell. The receiver cell filled with phosphate buffered saline (PBS) (pH 7.4; 16 mL), and the prepared hydrogel (1.0 g) was applied to the donor cell. The cells were heated in a water bath at 32 °C, and the receiver solution (1.0 mL) was collected at predetermined time intervals. After collecting the solution, PBS (pH 7.4; 1.0 mL) was added to maintain sink condition. Drug concentrations in the solution were determined by HPLC.
Quantification of KPF, CMT, and CFThe concentration of the model drugs in receiver solution was determined using HPLC equipped with an UV-visible spectrophotometer (Shimadzu Corp., Kyoto, Japan) and a C18 column (YMC-Pack ODS-A; 100 × 4.6 mm2 I.D.; YMC Co., Ltd., Kyoto, Japan).
In the case of KPF, methanol: 0.057% phosphoric acid (40 : 30, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The injection volume was 20 µL, and detection was performed at 219 nm.26)
For CMT, PBS : acetonitrile (89 : 11, v/v) was used as the mobile phase. The other conditions were the same as those described above.27)
For CF, methanol : 0.099% phosphoric acid (70 : 30, v/v) was used as the mobile phase and detection was performed at 275 nm. The injection volume was the same as that for the other two drugs, 20 µL.28)
In this study, three drugs were selected as models for the skin permeability experiments: KPF, a nonsteroidal anti-inflammatory drug; CMT, a histamine H2 receptor antagonist; and CF, a centrally stimulated analgesic. These drugs were chosen because they reflect the properties of the drugs contained in transdermal formulations used in clinical and research. KPF has an MW of 254.28 and log P of 3.12, indicating a relatively high hydrophobicity.29) The log P of KPF is known to vary with pH, and it has been reported that its solubility depending on the increase in pH.30,31) KPF is a weak acid, and exists as a mixture of ionic and molecular forms in experimental condition. According to pH-partition theory, the molecular form dominantly permeates through the membrane, the ionic form is changed into the molecular form to maintain equilibrium. This dynamic equilibrium allows membrane permeation to continue even under conditions where the ionic form predominates. Additionally, KPF is classified as a Biopharmaceutics Classification System (BCS) Class II drug (low solubility, high permeability), and it was treated as a hydrophobic drug in this study.31) CMT has an MW of 252.34 and log P of 0.40, been existing as intermediate between hydrophilic and lipophilic.27) CF, with an MW of 194.19 and log P of −0.07, is a highly hydrophilic drug.32) When compared with other drugs, CF is characterized by highly hydrophilic, making it a suitable permeability model drugs for hydrophilic drugs. Furthermore, CF is widely used in skin permeability studies, and its data are useful for comparison and validation with other drugs.33,34)
Figure 2 shows the results of the skin permeation experiments for the model drugs using a hydrogel. Among the model drugs, CF exhibited the highest permeability, followed by KPF and CMT. Although the cumulative drug permeation of CF and KPF showed a steady state, the cumulative permeation of CMT remained almost constant, indicating an overall low permeability.
Each point represents the mean ± standard deviation (S.D.) (n = 3). Multiple comparison tests were performed. Statistical significance was indicated only where significant differences were observed: * p < 0.01, ** p < 0.05.
The permeation pathways of drugs through the skin can be broadly classified into transepidermal and transappendageal pathways.8) These two pathways play different roles in drug permeation depending on the physicochemical properties of the drug and the skin structure. The transepidermal pathway is further divided into the transcellular and intercellular pathways. The former refers to the pathway through which drugs pass directly through the corneocytes in the SC. This pathway alternately permeates both hydrophilic keratinocytes and hydrophobic intercellular lipids, and it has been inferred that few drugs can permeate this pathway. In contrast, the latter involves drugs passing through the lipid layers filling the spaces between corneocytes, primarily used by hydrophobic drugs.35) The transappendageal pathway involves drug permeation through skin appendages, such as hair follicles, sweat glands, and sebaceous glands. Although these structures account for only 0.1 to 1% of the total skin surface area, the drugs with highly hydrophilic or have not only small but also larger MW may utilize this pathway for easier permeation through these pores.35)
CF has a relatively low MW and a high hydrophilicity.32) Indeed, CF exhibited high permeability. It is likely that this type of drug predominantly permeates through the transappendageal pathway as compared with others.33,36) The characteristics of CF, such as its MW and hydrophilicity, may have enabled efficient utilization of this pathway. Moreover, permeation via the transepidermal pathway was achieved over time.
KPF is highly hydrophobic and primarily utilizes the transepidermal pathway, particularly the intercellular pathway.29,37) Although drug permeation through the intercellular pathway takes longer than through the transcellular pathway, once permeation begins, sustained and stable permeation is expected. In the experimental results, the increase in the cumulative permeation of KPF is thought to be due to its gradual penetration through the intercellular lipids of SC.38) Therefore, while the initial permeation of KPF was not as high as CF, the cumulative permeation increased over time.
Although the MW of CMT was almost the same as that of KPF, it exhibited intermediate characteristics between being hydrophilic and hydrophobic.27) Although the difference in MW between CF and CMT is about 60, their functional group differs, which affects their hydrophobicity. As a result, this leads to a significant difference in permeability. Our experimental results showed that the cumulative permeation of CMT was not high. Due to the hydrophilicity/hydrophobicity of CMT, it may not have been able to effectively permeate through the transcellular pathway of SC, instead primarily utilizing the transappendageal pathway.32) Given that the transappendageal pathway constitutes only a small percentage of the total skin surface area,33,36) the overall permeability of CMT is not high. Additionally, the low cumulative permeation of CMT may be due to its insufficient distribution in the lipid layers of SC.
Drug permeation through SC relies heavily on diffusion through the lamellar structure of intercellular lipids. Diffusion pathways within the lamellar structure can be classified into two main types: free volume diffusion, which occurs in the vertical direction, and lateral diffusion, which occurs in the horizontal direction in the lipid lamellar structure.39) In regard to CF, due to its high hydrophilicity, it is likely that free volume diffusion was the primary pathway utilized within the lamellar structure. Free volume diffusion avoids the hydrophobic regions of the lamellar structure and allows drugs to travel vertically through tiny spaces in the lipid layers.40) The low MW of CF may also have contributed to its rapid permeation through this pathway. The large cumulative permeation of CF observed in the experimental results can be attributed to the rapid and efficient permeation facilitated by free volume diffusion.
In the case of KPF, which is highly hydrophobic, it is believed that it primarily advances through lateral diffusion within the lamellar structure.37) During lateral diffusion, the drug moves horizontally through the lipid layers of the lamellar structure, traversing the hydrophobic regions of the lipids.41) This could explain the large cumulative permeation of KPF. Hydrophobic drugs benefit more from lateral diffusion than from free volume diffusion, and these characteristics likely influence the skin permeability of KPF.
Regarding the low permeability of CMT, it is possible that its distribution into the lipids of the lamellar structure was inadequate, preventing it from efficiently utilizing either free volume diffusion or lateral diffusion.42) Although free volume diffusion is the primary pathway for hydrophilic molecules and progresses vertically through the lamellar structure, the overall hydrophobic nature of SC may have restricted the CMT permeation. Furthermore, because CMT is not highly hydrophobic, it may have been hindered by lateral diffusion, resulting in its overall low permeability.
In summary, CF exhibited high and stable permeability owing to its high hydrophilicity, low MW, efficient permeation via the transappendageal pathway, and free volume diffusion within the lamellar structure. KPF, which is highly hydrophobic, gradually permeates through the intercellular pathway over time and utilizes lateral diffusion within the lamellar structure, contributing to its skin permeability. CMT, possessing intermediate characteristics compared with the other two drugs, was unable to effectively utilize either the transappendageal or intercellular pathways, free volume, or lateral diffusion, resulting in low permeability. These results suggest that the physicochemical properties of drugs significantly influence the selection of skin permeation pathways.
The Skin Permeation Enhancement of Drugs by AlcoholWe focused on how the different alcohols affected the skin permeability of the drugs. In this study, we used three types of alcohols: EtOH (MW = 46.07, log P=−0.31),24) a representative short-chain alcohol commonly used as a formulation component in transdermal drug delivery; IPA (MW = 60.10, log P = 0.05),25) which exhibits amphiphilic properties due to its hydroxyl group and isopropyl group; and NPA (MW = 60.10, log P = 0.25),24,25) an isomer of IPA, used for comparison because of its differences in molecular structure. Figure 3 shows the results of skin permeability experiments using hydrogels formulated with 20 and 40% concentrations of EtOH, IPA, and NPA, respectively.
The control is the result of Fig. 1. Each point represents the mean ± S.D. (n = 3). Similar to Fig. 2, multiple comparison tests were performed. Statistical significance was indicated only where significant differences were observed: * p < 0.01, ** p < 0.05.
For KPF, NPA exhibited higher permeation in both 20% and 40% concentrations. Both IPA and EtOH exhibited similar permeation levels (Figs. 3(a), (b)). This order corresponds well with the hydrophobicity of the alcohols. Because KPF is more hydrophobic than the other drugs, its solubility likely increased in accordance with the hydrophobicity of the alcohols, resulting in a higher permeation. For CMT, similar to KPF, permeation was higher for NPA. IPA and EtOH exhibited similar permeation levels (Figs. 3(c), (d)). Because CMT has intermediate properties between hydrophilic and hydrophobic, the difference in log P values between EtOH and IPA had little effect on the skin permeation of CMT. This phenomenon was also observed for skin permeation of KPF. For CF, unlike the previous two drugs, IPA and EtOH exhibited higher permeation. Additionally, differences were observed depending on the alcohol concentration (Figs. 3(e), (f)). In the case of IPA, lower alcohol concentration resulted in increased permeation. Because CF is hydrophilic, the permeation-enhancing effect was greater with the more hydrophilic EtOH. In contrast, the addition of hydrophobic NPA led to decrease in permeation, which was not observed for the other two drugs. This suggests that in the initial stages of drug permeation, the CF hydrogel (control) may have utilized the transappendageal pathway more efficiently than the CF hydrogel (alcohol).
EtOH significantly increased the skin permeability of CF and enhanced the permeability of KPF and CMT. Increasing the permeability of low MW hydrophobic drugs through intercellular pathways is of great importance for formulation development. IPA enhanced the skin permeability of KPF and CMT more than EtOH, but the enhancement effect on CF was found to be smaller. NPA notably increased the skin permeability of KPF and CMT; however, the skin permeability of CF was lower than that of the control. Despite CF having the highest intrinsic skin permeability among the drugs tested, the skin permeability of KPF increased more in hydrogel (alcohol) than in the CF hydrogel. This suggests that in combination with KPF, these alcohols may strongly disrupt the order of the hydrocarbon chains in the intercellular lipids of SC, which is the main pathway for KPF.37,38) This effect was particularly pronounced with NPA, suggesting that the hydrophobicity of the alcohol itself may influence the extent of its impact on the hydrocarbon chain region.43)
Furthermore, while EtOH had the smallest effect on promoting KPF skin permeability, it had the greatest effect on CF skin permeability. This suggests that EtOH strongly affects the polar regions through which the CF pass during free volume diffusion. However, NPA did not enhance CF skin permeability, indicating that NPA may not affect the permeation pathway of CF. Although NPA may significantly affect the hydrocarbon chain region of lipids, it is likely to have a smaller impact on the polar regions.
The above findings can also be examined through structural analyses of lipid packing. For example, studies using IR spectroscopy have evaluated lipid disorder by measuring the stretching vibrations of lipid hydrocarbon chains in IR spectroscopy.44) Specifically, the C–H stretching vibrations (around 2800–3000 cm−1) indicate the degree of order in the lipid hydrocarbon chains. It has been reported that alcohols induce irregularities in the C–H stretching vibrations of lipid hydrocarbon chains, disrupting their order. Thermal analysis of SC lipids using differential scanning calorimetry revealed a decrease in phase transition temperature and a reduction in melting enthalpy due to the influence of alcohols. These results imply that the lipid layers loosen and their order collapses.44) In addition, X-ray diffraction analysis of changes in the lamellar structure of SC lipids showed an increase in interlayer spacing, reflecting the disruption of the lamellar structure by alcohols.44) This provides evidence that alcohol disrupts the structural integrity of lipid layers by inducing irregularities in lipid packing.
In the view of interaction between model drugs and alcohols, there exists some papers. EtOH is widely used as a representative chemical penetration enhancer, primarily due to its ability to increase in drug solubility, and alter the lipid bilayer of the SC. On the other hand, high concentrations of EtOH (≥50%) have been reported to enhance drug solubility and modify the partition coefficient within the SC.45) Additionally, a study by Moser et al. reported that the increase in skin permeability caused by EtOH is not due to a direct interaction with the drug but rather to its effects on the lipid structure of the SC.46)
Considering the multifaceted effects of alcohol on skin permeability, including its role in enhancing drug solubility and its direct action on the skin surface, quantifying all contributing factors comprehensively remains challenging. However, the Quantitative Structure–Activity Relationship (QSAR) approach may provide a viable means to achieve this in the future. By analyzing the correlation between the physicochemical properties of alcohols (e.g., log P, and molecular volume) and skin permeability coefficient (Kp), the impact of alcohols’ structural characteristics on skin barrier function has been quantitatively evaluated.47) Additionally, lower alcohols have been reported to swell the lipid bilayer and weaken the barrier function, thereby increasing drug permeability. This effect is dependent on the type and concentration of alcohol use.48) Moreover, drug permeability is also influenced by its physicochemical properties, such as log P, molecular weight, and solubility. Using QSAR analysis, the Kp value under different alcohol environments has been predicted.49) Furthermore, the extent of the permeation-enhancing effect can be evaluated through regression analysis based on QSAR models (e.g., log P vs. Kp). A study by Ghafourian et al. reported the prediction of skin permeability using QSAR techniques.50) From these perspectives, QSAR analysis enables a quantitative discussion of the permeation mechanisms. However, further experimental data and additional research are needed to strengthen these findings.
Changes in the Skin Permeation Enhancement Effect with Alcohol ConcentrationThe drug permeation rate (flux) was evaluated for 20 and 40% alcohols (EtOH, IPA, and NPA) to investigate the difference in the skin permeation enhancement effect of drugs as a function of alcohol concentration (Fig. 4). When determining the flux value, data points from 6 to 12 h were used (pseudo-steady state).
The values were calculated at 20% (left) and 40% (right) alcohols, respectively. Each column represents the mean ± S.D. (n = 3). T-tests were performed. Statistical significance was indicated only where significant differences were observed: * p < 0.01, ** p < 0.05.
For hydrophobic KPF, the skin permeation enhancing effect was significant when hydrophobic NPA was used. This effect was more pronounced as the amount of NPA increased. A similar trend was observed for CMT, although the permeation amount was significantly higher for KPF. In contrast, for hydrophilic CF, the enhancement effect was greater with hydrophilic EtOH. However, as the concentration of EtOH increased, the permeation decreased. A similar tendency was observed with IPA. Alcohol enhances permeability by altering membrane fluidity; however, at higher concentrations, it can destabilize the structure, emphasizing a more hydrophobic environment. This phenomenon may reduce the efficiency of hydrophilic molecules, such as CF, in permeating through the membrane. Consequently, the permeation rate may decrease beyond a certain alcohol concentration.51,52) Furthermore, CF is a highly hydrophilic drug, and its permeation behavior in the presence of alcohol has been reported to differ from other drugs. At high alcohol concentrations, excessive dehydration of the SC occurs, making it more difficult for hydrophilic drugs like CF to pass through the hydrophilic pathways of the SC.52)
These results indicated that the effects of concentration changes were not uniform, even for the same alcohols. The results also suggest that there may be alcohols that can alter the skin permeability of each drug in a concentration-dependent manner, such as NPA for KPF and CMT, and IPA for CF.
On the other hand, for the hydrophilic drug CF, the hydrophilic EtOH exhibited a greater permeation-enhancing effect. However, as the amount of EtOH increased, the permeation decreased. A similar tendency was observed with IPA. These results suggested that even with the same alcohol, the effects of concentration changes are not uniform.
Even well-known short-chain alcohols such as EtOH, IPA, and NPA clearly show that each has specific drugs that are more suitable in formulation. Although EtOH, IPA, and NPA are classified as short-chain alcohols with only slight structural differences, their effects on drug skin permeation are not uniform and their concentration-dependent effects vary. The permeation enhancement of KPF by alcohols may be influenced by the log P value of the alcohol itself, with higher concentration leading to greater enhancement. For hydrophobic drugs such as KPF and CMT, which have MWs of approximately 250, an increase in the log P value of alcohol resulted in enhanced skin permeation. In contrast, for hydrophilic drugs such as CF with an MW of approximately 150, permeation was enhanced when the log P value of alcohol was lower, differing from KPF and CMT.
This work was supported by JSPS KAKENHI (Grant Nos. JP20K08699 and JP23K07775).
The authors declare no conflict of interest.
