http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.954727
RESEARCH ARTICLE
Skin deposition and permeation of finasteride in vitro: effects of propylene glycol, ethanol and sodium lauryl sulfate Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Nyu Medical Center on 04/16/15 For personal use only.
Ekapol Limpongsa1, Napaphak Jaipakdee1,2, and Thaned Pongjanyakul1 1
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand and 2Center for Research and Development of Herbal Health Products, Khon Kaen University, Khon Kaen, Thailand Abstract
Keywords
The objective of this study was to investigate the effects of propylene glycol (PG), ethanol (EtOH) and sodium lauryl sulfate (SLS) on the in vitro deposition and permeation of finasteride (FNS). A side-by-side diffusion cell mounted with a pig ear skin and a saturated solution of FNS in PG (10, 20% v/v), EtOH (10, 20% v/v) or SLS (0.5, 1% w/v) vehicles were used. Incorporation of PG, EtOH or SLS caused a significant increase in FNS solubility both in the solution and on the skin with SLS4EtOH4PG. The results obtained from skin deposition studies showed that the FNS deposition rate and time increased in the same order as that of the solubility. The deposition kinetics of FNS solubilized in PG, EtOH and SLS vehicles followed either zero-order, square-root-of-time or pseudo-first-order kinetic models depending on the type and concentration of the enhancer. The permeation studies demonstrated that FNS permeation fluxes were enhanced only by EtOH vehicles. These results suggest that PG and SLS could be used as deposition enhancers, while EtOH could be the effective permeation enhancer of FNS. The obtained results can be used as the considerable insights for formulating the topical and transdermal products of FNS.
Enhancer, finasteride, permeation, skin deposition, solution
Introduction Finasteride (FNS) is an inhibitor of the human 5a-reductase (5a-R) enzyme, which inhibits the conversion of testosterone to dihydrotestosterone (DHT)1,2. There are two types of 5a-R enzymes present in human tissues, referred to as Type 1 and Type 2. In the skin of the scalp, Type 1 5a-R is located in the sebaceous glands, whereas Type 2 5a-R is located in the connective tissue sheaths and dermal papillae of the hair follicles. It has been postulated that the activity of Type 2 5a-R is the cause of androgenetic alopecia (AGA) or male pattern baldness. FNS inhibits Type 2 5a-R more effectively than Type 1 5a-R. Oral FNS was approved by the US FDA in 1997 for the treatment of AGA in men only1. Upon long-term oral administration of FNS, the levels of DHT found in bald scalps were decreased3. FNS is well tolerated and has a favorable adverse event history. The most common adverse events included decreased libido, sexual dysfunction and decreased ejaculate volume resulting in the discontinuation of FNS therapy1. Many studies have reported that the therapeutic effect of FNS solutions and gels included decreased hair loss in both men and women4, regression of sebaceous glandular and ductal hyperplasia in fuzzy rats5 and the re-enlargement of hair follicles in human scalp skin grafts transplanted onto SCID mice2. These studies
Address for correspondence: Ekapol Limpongsa, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Tel: +66 43 362092. Fax: +66 43 362092. E-mail: ekapol@ hotmail.com
History Received 4 July 2014 Revised 3 August 2014 Accepted 5 August 2014 Published online 27 August 2014
encourage the use of FNS via topical application. Moreover, it is more favorable to use a percutaneous approach for the delivery of therapeutic agents directly onto the targeted tissues to reduce first-pass effects and systemic side effects6. Drug transport of a formulation to the systemic circulation is a multistep process which involves (a) dissolution within the formulation and the drug’s subsequent release, (b) partitioning of the drug into the stratum corneum (SC) of the skin, (c) diffusion through the SC, (d) partitioning of the drug from the SC into the aqueous viable epidermis, (e) diffusion through the viable epidermis and into the upper dermis and (f) uptake into the local capillary network and eventually into the systemic circulation7. Human skin is an effective barrier to chemical permeation8. Many strategies have been suggested to overcome the low permeability of drugs through the skin. A popular approach is the use of permeation enhancers that interact with skin constituents to promote drug flux9. An effective permeation enhancer may increase the diffusivity of the skin, partitioning of the drug between the formulation and the skin or the effective concentration of the drug in the vehicle8. To administer FNS via a topical route, a preparation that improves skin deposition (penetration into skin) while reducing skin permeation (systemic absorption) is desirable10. Javadzadeh et al.11 reported that the permeation of FNS through rat skin decreased when cetyltrimethylammonium bromide or sodium lauryl sulfate (SLS) was used; unfortunately deposition was not measured. Many FNS vesicles including liposomes, niosomes, ethosomes and polymersomes have been previously studied6,10,12,13. FNS permeation flux values were 1.23-fold lower
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for liposomes10,13, 2.31-fold higher for ethosomes6 and 1.33- to 1.57-fold higher for polymersomes12 when compared to hydroethanolic solutions of FNS (30%). The hydroethanolic solution allowed for higher FNS skin deposition than liposomes; however, the deposition levels were lower than those obtained with ethosomes. In another study, liposomes enhanced FNS skin deposition; however, problems related to chemical and microbial stability were encountered and thus limit their applications14. The objective of this study was to investigate the effects of propylene glycol (PG), ethanol (EtOH) and SLS on skin deposition and permeation of FNS in vitro. The in vitro skin deposition kinetics were also studied. Skin deposition was defined as the amount or concentration of drug deposited in the skin15. PG and EtOH are inexpensive, nontoxic, and well-tolerated co-solvents, whereas SLS is an anionic surfactant that can penetrate and interact strongly with the skin. Their efficacy and action have been reported in the literature16–21. The results obtained from this study can be used as the insight information for formulating the topical solution of FNS.
(PBS) at 32 C for 15 min and was then measured the thickness using a dial thickness gauge (PeacockÕ , Labtek, Scotts Valley, CA). The hydrated skin was mounted between the donor and receptor compartments with a clamp and was further hydrated with PBS for 45 min. The saturated solution of FNS (3 mL) was added to the donor compartment, which was in contact with the SC side of the skin. The receptor compartment was filled with 3 mL of 40% v/v PEG400 in deionized water. After 0.5, 1, 2, 3, 4 or 10 h, the skin samples were removed and washed on both sides with 0.5 mL of water (3 times) followed by 0.5 mL of methanol (3 times). The part of each skin that directly contacted the saturated solution of FNS (area of 0.694 cm2) was separated using scissors and dried at 50 C for 24 h. The dried skin was cut into small pieces, and FNS was extracted with methanol. The extraction was performed twice. The samples were analyzed for FNS content by HPLC. The deposition amount of FNS per unit area (Qs; mg/cm2) was plotted against time (h).
Materials and methods
The saturated deposition amount per unit area (Qs,sat; mg/cm2) was considered the average deposition amount of the plateau portion of the deposition profile. The volume of skin was calculated by multiply the diffusion area with the skin thickness. The deposition concentration was the deposition amount per volume of skin (Cs; mg/mL). The saturated deposition concentration in the skin or the skin solubility (Cs,sat) was the average deposition concentration of the plateau portion on the deposition profile. The deposition time to saturation (Tdep; h) was the time Qs,sat obtained from the deposition profile. The partition coefficient of FNS from the vehicle onto the skin (K) was obtained using the saturated concentration in the vehicle (Cv,sat or solubility in vehicle) and the saturated concentration in the skin (Cs,sat or solubility in skin) as shown in Equation (1)7.
Materials Finasteride (FNS) was purchased from Aurisco Pharmaceutical, Shanghai, China. Propylene glycol (PG) and sodium lauryl sulfate (SLS) were obtained from S. Tong Chemicals, Bangkok, Thailand. Ethanol (EtOH) and methanol were purchased from QReC, New Zealand. Polyethylene glycol 400 (PEG400) was purchased from Merck KGaA, Darmstadt, Germany. Deionized water was used throughout the studies. All chemicals were of reagent or high-performance liquid chromatography (HPLC) grade. Methods
Data analysis
Solubility study Excess amounts of FNS were added to each solvent. The mixture was sonicated for 1 h and then equilibrated at 32.0 ± 0.5 C in a shaking water bath (Digital Temperature Controller, Polyscience Inc, Warrington, PA) for 72 h. Mixtures were then filtrated through a membrane filter (0.45 mm, 13 mm, Millipore filter, Millipore, Billerica, MA). The filtered solution was then diluted and analyzed for FNS content by HPLC assay at 210 nm as described below. Skin preparation Porcine ears were obtained from a local slaughter house and cleaned with water. After soaking the ears in water at 60 C for 45 s, the intact epidermis was peeled off with forceps, washed with water and kept at 20 C until use (within 4 weeks)22. The frozen skin was thawed at an ambient temperature before use.
K ¼
ð1Þ
Kinetics of deposition The saturated solutions were used in this study to ensure that the drug is at its maximum thermodynamic activity. During the deposition process, it is thought that the rate is proportional to the difference between the amount of deposition at time t and the deposition capacity or saturated deposition amount of drug in the skin (Qs,sat Qs)23. The zero-order, square-root-of-time and pseudo-first-order kinetic models were used to interpret the experimental kinetic data and are represented below by Equations (2)–(4) [or 5], respectively23–25,
In vitro skin deposition study Saturated solution preparation. Saturated solutions of FNS were prepared by sonicating the mixture of excess FNS and solvent (1 h) and then equilibrating at 32.0 ± 0.5 C (72 h). This mixture was used for the deposition and permeation studies without any further filtration. The in vitro deposition of FNS from the saturated solution into the pig ear skin was conducted using a side-by-side diffusion cell with a diffusion area of 0.694 cm2 (Crown Glass Company, Somerville, NJ). The system was connected to a water bath maintained at a temperature of 32.0 ± 0.5 C. A thawed skin was hydrated by immersing in a pH 7.4 phosphate buffered saline
Cs, sat Cv, sat
or
Qs, sat Qs ¼ k0 t
ð2Þ
Qs, sat Qs ¼ ksq t1=2
ð3Þ
Qs, sat Qs ¼ Qs, sat expk1 t
ð4Þ
lnðQs, sat Qs Þ ¼ lnðQs, sat Þ k1 t
ð5Þ
where Qs is the deposition amount of the drug at time t, Qs,sat is the saturated amount of drug in the skin and k0, ksq and k1 are the deposition rate constants of the zero-order, square-root-of-time and pseudo-first-order kinetic models, respectively.
Skin deposition and permeation of finasteride in vitro
DOI: 10.3109/10837450.2014.954727
The deposition rate constants (k0, ksq and k1), the determination coefficient (R2) and the calculated saturated amount of drug in the skin (Qs,sat,cal) were obtained by fitting the kinetic models to the experimental data. The experimental data were compared with the predicted curves using zero-order, square-root-of-time and pseudofirst-order models by using the normalized standard deviation [Equation (6)]23 as defined by sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 P ðQs, exp Qs, cal Þ=Qs, exp ð6Þ Dqð%Þ ¼ 100 N 1
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where the subscripts exp and cal denote the experimental and calculated values, respectively, and N is the number of data points. In vitro skin permeation study The in vitro permeation of FNS from the saturated solution through the pig ear skin was conducted using a side-by-side diffusion cell under conditions similar to those used in the skin deposition studies. At predetermined times, 1.0-mL samples were taken from the receptor compartment and equal volumes of 40% PEG400 in deionized water were immediately added after each sampling. The concentration of FNS was analyzed by HPLC. The cumulative amount of drug that permeated the skin was plotted against time. Data analysis The steady-state flux (Jss), the permeability coefficient of the skin (Ps) and the diffusivity of the skin (Ds) are defined by Equations (7)–(9)26. Jss ¼ Ps Cv, sat ¼
K Ds Ds Cs, sat Cv, sat ¼ L L
ð7Þ
3
consisted of methanol: water at a volume ratio of 75:25. The retention time of FNS was approximately 7 min. The standard curve was linear over a concentration range of 0.8–10 mg/mL with an R2 value 4 0.99. The day-to-day relative standard deviations (RSD) for this assay were less than 5%. Statistical analysis Each experiment was repeated at least three times. The results are expressed as the mean ± S.D. One-way analysis of variance was used to test the statistical significance of differences among groups. Statistical significance of the differences of the means was determined by Student’s t-test. All statistical tests were run using the SPSS program for MS Windows, release 19 [SPSS (Thailand) Co. Ltd., Bangkok, Thailand]. The significance was determined with 95% confident limits ( ¼ 0.5) and was considered significant at a level of p less than 0.05.
Results FNS solubility in the vehicle The effects of PG (10 and 20%), EtOH (10 and 20%) and SLS (0.5 and 1.0%) on FNS solubility (at 32 C) were determined and are reported in Table 1. The solubility of FNS in water was 42.3 ± 1.9 mg/mL. Addition of co-solvents (PG or EtOH) significantly improved FNS solubility. These solubility improvements were significantly pronounced at higher concentrations of co-solvent (p50.05). The solubility of FNS in different solvents occurred in the following order: 20% EtOH420% PG410% EtOH410% PG, respectively. Addition of SLS could also increase the solubility of FNS; the solubility was dramatically increased approximately 32- and 68-fold when 0.5 or 1.0% SLS was used, respectively. FNS skin deposition
Ps ¼
K Ds L
ð8Þ
Ds ¼
Jss L Cs, sat
ð9Þ
where L is the thickness of skin. HPLC analysis FNS content was determined by an HPLC system (Perkin-Elmer, Waltham, MA) consisting of a UV detector (model 1022 LC plus) and a pump (series 200 LC). The chromatographic separation was achieved on a Hypersil Gold C-18 column (250 4.6 mm, 5 mm; Thermo Electron Corporation, Waltham, MA) with a flow rate of 1 mL/min and UV detection at 210 nm. The mobile phase
The amount of FNS deposited in skin (Qs) was plotted against time as shown in Figure 1. It could be seen that the FNS skin deposition profiles obtained from the PG, EtOH and SLS solutions were higher than that of the control solution. SLS resulted in the highest FNS deposition as compared to those obtained from EtOH and PG, respectively. The increase in PG, EtOH or SLS concentrations in the vehicle resulted in higher skin deposition levels. Table 1 reports the skin solubility (Cs,sat), partition coefficient (K), saturated deposition amount (Qs,sat) and deposition time (Tdep) of FNS in various vehicles. The addition of PG, EtOH or SLS resulted in an increase in skin solubility and saturated skin deposition levels. The increase in PG, EtOH or SLS concentration resulted in a significant increase in solubility and deposition amount of FNS in skin (p50.05). The ranked order of FNS skin solubility and deposition amounts are the same as the vehicle solubility (Cv,sat) rank.
Table 1. Effects of propylene glycol (PG), ethanol (EtOH) and sodium lauryl sulfate (SLS) on the vehicle solubility (Cv,sat), the skin solubility (Cs,sat), the partition coefficient (K), the saturated deposition amount (Qs,sat) and the deposition time to saturate (Tdep) of finasteride from saturated solution in pig ear skin at 32 C (mean ± SD, n ¼ 3). Vehicles
Cv,sat (mg/mL)
Cs,sat (mg/mL)
K
Qs,sat (mg/cm2)
Tdep (h)
Water (control) 10% PG 20% PG 10% EtOH 20% EtOH 0.5% SLS 1.0% SLS
42.3 ± 1.9 89.9 ± 5.2a 202.5 ± 4.1a 124.2 ± 6.5a 362.4 ± 8.8a 1360.3 ± 43.2a 2869.4 ± 83.2a
61.0 ± 6.0 104.9 ± 14.3a 177.1 ± 32.0a 148.6 ± 11.8a 335.8 ± 53.9a 718.3 ± 57.5a 1237.6 ± 67.6a
1.5 ± 0.1 1.2 ± 0.2a 0.9 ± 0.2a 1.2 ± 0.1a 0.9 ± 0.2a 0.5 ± 0.0a 0.4 ± 0.0a
9.5 ± 0.1 16.7 ± 0.8a 26.6 ± 1.1a 21.9 ± 1.3a 59.1 ± 3.0a 114.8 ± 1.6a 209.3 ± 1.0a
1.9 3.4 3.4 3.8 4.0 3.0 3.0
a
Significant different from control at p50.05.
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The partition coefficient can be calculated based on vehicle and skin solubility according to Equation (1). Surprisingly, the addition of PG (10%), EtOH (10%) or SLS (0.5%) led to a significant decrease in the partition coefficient when compared to the control. Increasing the PG or SLS concentration had no further effect on the partition coefficient. However, the partition coefficient of FNS significantly decreased (p50.05) as EtOH concentration increased (10–20%). The deposition time to saturation (Tdep) was the time that reached the saturated deposition amount per unit area, obtained from the deposition profile. It should be noted that there was no lag time for deposition of all vehicles used in this study. As can be seen in Table 1, the deposition times of FNS in all vehicles containing permeation enhancers were longer than those of the control and ranged from 3.0 to 4.0 h. EtOH vehicles yielded the longest deposition time as compared to those obtained from the others.
and the normalized standard deviation (Dq) for all vehicles. The examples of the deposition kinetics of FNS in water, 20% PG and 0.5% SLS solutions obtained from different kinetic models are depicted in Figure 2. It is obvious from the rate constants that the deposition process was accelerated by the incorporation of PG, EtOH or SLS into the vehicle. This indicates that the deposition of FNS from the vehicles containing these enhancers onto the skin was more rapid and favorable than water alone. The effect on the rate of deposition was more pronounced when higher concentrations of PG, EtOH or SLS were used. Considering that the best-fit model shall yield the high R2 together with the low Dq values, the FNS deposition process from water, 10% PG and 20% EtOH vehicles followed the squareroot-of-time model (Table 2). In contrast, the FNS deposition process in 20% PG and 10% EtOH vehicles followed the pseudofirst-order model, whereas in the vehicle containing SLS followed the zero-order model.
Deposition kinetics
FNS skin permeation
The kinetic provides valuable insights of the deposition mechanism involved. In this study, the zero-order, square-root-of-time and pseudo-first-order kinetic models [Equations (2), (3) and (5), respectively] were selected to fit the deposition kinetic data. Table 2 shows the values of the rate constants obtained from each kinetic model (k0, ksq and k1), the determination coefficient (R2), the calculated saturated deposition amount of FNS, Qs,sat,cal
In vitro permeation of saturated FNS solutions through the pig ear skin was studied and the permeation-time profiles are shown in Figure 3. The permeation profiles of FNS in PG solutions (10 and 20%) and SLS solutions (0.5 and 1.0%) were comparable to those of the control vehicle (water), while profiles from EtOH solutions (10 and 20%) were higher than those of the control. Table 3 shows the corresponding steady-state flux (Jss), permeability coefficients
Figure 1. Effects of vehicle types (Water, PG, EtOH and SLS) on skin deposition amount (Qs) versus time plots of FNS from saturated solution (n ¼ 4).
Table 2. Deposition rate constants (k0, ksq and k1) and saturated deposition amount (Qs,sat) of finasteride from various kinetic models. Zero-order Vehicles Water (control) 10% PG 20% PG 10% EtOH 20% EtOH 0.5% SLS 1.0% SLS
Square root of time
Pseudo-first-order
k0 (mg/cm2/h)
R2
Qs,sat,cal (mg/cm2)
Dq (%)
ksq (mg/cm2/h1/2)
R2
Qs,sat,cal (mg/cm2)
Dq (%)
k1 (h1)
R2
Qs,sat,cal (mg/cm2)
Dq (%)
3.94 5.74 7.95 9.07 19.70 42.15 70.48
0.856 0.972 0.964 0.897 0.839 0.998 0.997
8.1 15.8 25.1 19.1 51.1 113.1 206.1
36.0 19.5 21.8 31.1 37.1 6.8 8.4
6.06 8.25 11.52 13.72 30.69 58.33 97.47
0.985 0.978 0.985 0.999 0.990 0.930 0.928
9.4 17.2 27.2 21.8 57.8 121.7 220.5
10.9 15.8 16.6 4.6 13.3 34.6 32.1
1.05 1.08 0.48 1.13 0.62 0.70 0.59
0.986 0.939 0.997 0.998 0.955 0.978 0.980
8.8 14.7 29.4 22.1 52.2 124.6 221.2
17.1 24.6 11.1 4.3 31.9 29.8 21.8
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Figure 2. Deposition kinetics of FNS from (a) water, (b) 20% PG and (c) 0.5% SLS solution in pig ear skin.
(Ps), skin diffusivities (Ds) and lag times (Tlag) of FNS permeations. It can be seen that PG and SLS had an insignificant effect on the flux. In contrast, EtOH resulted in a significantly higher flux when compared to the control. Increase in EtOH concentration from 10% to 20% resulted in significant increases of the FNS flux (p50.05). The permeability coefficient of FNS through the pig ear skin decreased significantly when PG, EtOH or SLS was added to the chamber. The increasing PG, EtOH or SLS concentrations in the vehicle led to significant decreases in the permeability coefficients (p50.05). PG, EtOH or SLS had insignificant effect on the lag time when compared to the control. However the skin diffusivity which was calculated using Equation (9) decreased significantly when the
vehicle contained PG, EtOH or SLS. Interestingly, increase the EtOH concentration from 10% to 20% significantly increased the skin diffusivity.
Discussion FNS is a lipophilic drug with low solubility in water and a high log Poct value27. The improvement of FNS solubility by PG and EtOH was attributed to their co-solvent effects on the polarity of the vehicle. EtOH resulted in higher solubility improvements when compared to those obtained from PG. This effect could be attributed to the lower polarity of EtOH (solubility parameter, , of 12.74 cal1/2 cm3/2) when compared to PG ( of 14.76 cal1/2 cm3/2)28. Nevertheless, the highest solubility increase was
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Figure 3. Effects of vehicle types (a: PG; b: EtOH; c: SLS) on cumulative permeation-time profiles of FNS from saturated solution (n ¼ 4).
Table 3. Effects of propylene glycol (PG), ethanol (EtOH) and sodium lauryl sulfate (SLS) on the steady-state flux (Jss), the permeability coefficient (Ps), the skin diffusivity (Ds) and the lag time (Tlag) of finasteride from saturated solution across pig ear skin at 32 C (mean ± SD, n ¼ 4).
Vehicles Water (control) 10% PG 20% PG 10% EtOH 20% EtOH 0.5% SLS 1.0% SLS a
Ps Jss (mg/cm2/h) (10 3 cm/h) 2.8 ± 0.6 2.8 ± 0.5 3.2 ± 0.3 3.9 ± 0.5a 8.0 ± 0.8a 2.0 ± 0.1 2.4 ± 0.3
67.0 ± 13.5 31.3 ± 6.0a 15.9 ± 1.7a 31.7 ± 4.3a 22.1 ± 2.3a 1.5 ± 0.1a 0.8 ± 0.1a
Significantly different from control at p50.05.
Ds (103 cm2/h)
Tlag (h)
7.1 ± 1.1 4.3 ± 0.9a 2.9 ± 0.3a 3.7 ± 0.7a 4.9 ± 0.2a 0.4 ± 0.0a 0.3 ± 0.1a
1.7 ± 0.5 2.3 ± 0.5 2.4 ± 0.2 2.1 ± 0.1 2.2 ± 0.5 2.0 ± 0.5 2.6 ± 0.5
observed for the SLS solution, which could be due to the effects of micellar solubilization. The critical micelle concentration (CMC) of SLS is only 0.24%29. In vitro skin deposition and permeation of saturated FNS solutions was studied using a side-by-side diffusion cell. A 40% PEG400 solution was used as a receiver medium in order to maintain the sink condition throughout the experiment30. PEG400 is one of the few solvents widely used to increase the solubility of the investigated drug in the receiver fluid. Depends on the hydrophobicity of the investigated compounds, various concentrations of solvent(s) were used22,31,32. In the case of FNS, it was found that the solubility of FNS in 40% PEG400 solution was sufficient to obtain the sink condition. The solubility of FNS at 32 C in 40% PEG400 solution (786.6 ± 5.3 mg/mL) was 19-fold greater than it was in PBS (540 mg/mL). It has been
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demonstrated that the incorporation of up to 40% v/v PEG400 does not influence the barrier properties of SC33. The transdermal/topical delivery is a complex phenomenon governing with the release from the vehicle, the enhancing potency of vehicle and partitioning of drug into the skin. The release of drug from a vehicle into the skin and its diffusion across the skin are controlled by physicochemical factors sensitive to the molecular properties of the drug, the vehicle and the skin34. The delivery process can be affected by interactions that occur between the drug and skin, vehicle and skin, drug and vehicle, and drug, vehicle and skin. Increasing a drug’s solubility in the vehicle leads to larger concentration gradient between the vehicle and the skin. This idea may be responsible for the increases observed in the deposition rate of FNS. PG and EtOH may deposit onto the skin17,21,35,36 whereas SLS may disrupt the SC layer of the skin37,38, resulting in a decrease in the barrier property of the skin and subsequently enhance FNS solubility and deposition rate. The higher affinity of drug to the vehicle could be the reason for the decreases in FNS partition coefficient. Addition of PG or EtOH to water rendered the vehicle less polar. The affinity of FNS dissolved in the vehicle increased with increasing ratios of PG or EtOH. As a result, the partition coefficient decreased. The relationship between the solubility parameters (indicating polarity) and the partition coefficient with a high coefficient of determination is shown in Figure 4. Some of the PG or EtOH from the vehicle could also partition, diffuse and deposit on the skin17,19. EtOH has a lower molecular weight but a greater log Poct value than PG27. Therefore, EtOH has a higher affinity for the skin than PG. The higher amounts of EtOH may be allocated for deposition onto the skin when compared to PG. This phenomenon might cause the longer deposition times of FNS in the EtOH vehicle when compared to that in the PG vehicle. The skin deposition process of FNS from 10% PG and 20% EtOH vehicles followed the square-root-of-time model, indicating the limitation of FNS diffusion within the skin7,25. In contrast, the FNS deposition process in 20% PG and 10% EtOH vehicles followed the pseudo-first-order model, indicating that the transport may be controlled by a chemical reaction between FNS and the active sites of the SC in skin39. PG in the vehicle may extract some water from the SC19, leading to an increase in the SC barrier and a decrease in FNS diffusivity in the skin. At low concentrations, EtOH could increase the fluidity of
Skin deposition and permeation of finasteride in vitro
7
the lipoidal pathway in the SC19 and enhance FNS skin affinity. Therefore, the diffusivity in skin was decreased. As can be seen from Equation (8), the decrease of the diffusivity and partition coefficient in skin resulted in the decrease of the permeability coefficient of FNS from PG and EtOH vehicles. From Equation (7), permeation flux is a product of permeability coefficient and concentration of drug in the apply vehicle. In the case of PG vehicles, the slight increase in FNS solubility (concentration) in the vehicle might compensate the decrease in permeability coefficient; therefore, the FNS fluxes from the PG vehicles were not different from that of the control. However, enhancement of FNS flux from the EtOH vehicles may be the result of higher concentrations of FNS in the vehicles when compared to those from the PG vehicles and the control. Incorporation of SLS at concentrations above CMC caused the formation of micelles around FNS and the enhancement of FNS solubility in the vehicles. SLS can penetrate and strongly interact with the skin leading to the enhancement of the amount, solubility and time of FNS deposition in the skin16,40. However, FNS may have a high affinity for micelles; thus, the fraction of unassociated diffusing species was lower24. The partition coefficient of FNS was also very low. Because hydrophobic interactions of the SLS alkyl chain with the skin structure leaves an end sulfate group of the surfactant exposed, it creates additional sites in the membrane that can permit an increase in skin hydration37. The FNS skin deposition process from the vehicle containing SLS followed the zero-order model, indicating an increase in the number of deposition sites. The diffusion of FNS into the skin was dependent on the diffusion of micelles. SLS micelles exhibit sizes greater than FNS and can strongly interact with the skin; therefore, their diffusion was very slow. The skin diffusivity of 0.5% and 1.0% SLS solutions were approximately 18 and 24 times less than that of the control. The very low diffusivity and partition coefficient in skin caused the very low permeability coefficient of FNS from SLS vehicles. As compared to the control, the permeability coefficient of FNS from 1.0% SLS solution decreased for 84 times. Nevertheless, FNS fluxes from the vehicles containing SLS were not significantly different from that of the control. This is attributed to the effect of SLS on the FNS solubility in the vehicle. SLS is an anionic surfactant that is found in many therapeutic preparations9. It has been shown that SLS application disrupted the skin barrier function resulting in increased transdermal water loss41,42. The effect of SLS on excised human skin was previously studied and found that SLS at concentration up to 5% had no visible effects on tissue architecture when compared with untreated samples41. The report of Nokhodchi et al.18 which performed permeation experiments with rat skin of lorazepam as a function of SLS concentrations (0.5, 1.0, 2.5, 5.0% w/w) suggested that the extensive skin damage might occur when the concentration of SLS higher than 2.5%.
Conclusions
Figure 4. Relationship between solubility parameter and partition coefficient of FNS from water, PG and EtOH solutions (Y ¼ 0.3257x 6.1718, R2 ¼ 0.9934).
The effects of PG, EtOH and SLS on FNS skin deposition and permeation were studied. SLS (0.5 and 1%) yielded the highest solubility of FNS in the vehicles, and therefore skin deposition. In contrast, permeation studies demonstrated that only the EtOH containing vehicle (10 and 20%) could enhance FNS permeation fluxes. The results of this study suggest that EtOH could be the effective permeation enhancer; whereas, when the local effects are desirable, PG and SLS could be used as deposition enhancers of FNS. These results provided the considerable insights for selecting a suitable enhancer for transdermal or topical products of FNS. Nevertheless, further investigations regarding skin irritation effect of these vehicles shall be performed.
8
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Declaration of interest The authors wish to thank the Thailand Research Fund (MRG 5380119), the Commission of Higher Education and Khon Kaen University, Ministry of Education, Thailand, for financial support. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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References 1. Libecco JF, Bergfeld WF. Finasteride in the treatment of alopecia. Expert Opin Pharmacother 2004;5:933–940. 2. Sintov A, Serafimovich S, Gilhar A. New topical antiandrogenic formulations can stimulate hair growth in human bald scalp grafted onto mice. Int J Pharm 2000;194:125–134. 3. Drake L, Hordinsky M, Fiedler V, et al. The effect of finasteride on scalp skin and serum androgen levels in men with androgenetic alopecia. J Am Acad Dermatol 1999;41:550–554. 4. Mazzarella F, Loconsole F, Cammisa A, et al. Topical finasteride in the treatment of andrognic alopecia. Preliminary evaluations after a 16-month therapy course. J Dermatol Treat 1997;8:189–192. 5. Ye F, Imamura K, Imanishi N, et al. Effects of topical antiandrogen and 5-alpha-reductase inhibitors on sebaceous glands in male fuzzy rats. Skin Pharmacol 1997;10:288–297. 6. Rao Y, Zheng F, Zhang X, et al. In Vitro percutaneous permeation and skin accumulation of finasteride using vesicular ethosomal carriers. AAPS PharmSciTech 2008;9:860–865. 7. Kalia YN, Guy RH. Modeling transdermal drug release. Adv Drug Deliv Rev 2001;48:159–172. 8. Barry BW. Lipid-protein-partitioning theory of skin penetration enhancement. J Control Release 1991;15:237–248. 9. Williams AC, Barry BW. Penetration enhancers. Adv Drug Deliv Rev 2004;56:603–618. 10. Tabbakhian M, Tavakoli N, Jaafari MR, Daneshamouz S. Enhancement of follicular delivery of finasteride by liposomes and niosomes: 1. In vitro permeation and in vivo deposition studies using hamster flank and ear models. Int J Pharm 2006;323:1–10. 11. Javadzadeh Y, Shokri J, Hallaj-Nezhadi S, et al. Enhancement of percutaneous absorption of finasteride by cosolvents, cosurfactant and surfactants. Pharm Dev Technol 2010;15:619–625. 12. Caon T, Porto LC, Granada A, et al. Chitosan-decorated polystyrene-b-poly(acrylic acid) polymersomes as novel carriers for topical delivery of finasteride. Eur J Pharm Sci 2014;52:165–172. 13. Daneshamouz S, Tabbakhian M, Tavakoli N, Jaafari MR. Influence of liposomes and niosomes on the in vitro permeation and skin retention of finasteride. Iran J Pharm Sci 2005;1:119–130. 14. Biruss B, Valenta C. Skin permeation of different steroid hormones from polymeric coated liposomal formulations. Eur J Pharm Biopharm 2006;62:210–219. 15. Tuntiyasawasdikul S, Limpongsa E, Jaipakdee N, Sripanidkulchai B. Transdermal permeation of kaempferia parviflora methoxyflavones from isopropyl myristate-based vehicles. AAPS PharmSciTech 2014;15:947–955. 16. Borra`s-Blasco J, Lo´pez A, Morant MJ, et al. Influence of sodium lauryl sulphate on the in vitro percutaneous absorption of compounds with different lipophilicity. Eur J Pharm Sci 1997;5:15–22. 17. Herkenne C, Naik A, Kalia, YN, et al. Effect of propylene glycol on ibuprofen absorption into human skin in vivo. J Pharm Sci 2008;97: 185–197. 18. Nokhodchi A, Shokri J, Dashbolaghi A, et al. The enhancement effect of surfactants on the penetration of lorazepam through rat skin. Int J Pharm 2003;250:359–369. 19. Panchagnula R, Salve PS, Thomas NS, et al. Transdermal delivery of naloxane: effect of water, propylene glycol, ethanol and their binary combinations on permeation through rat skin. Int J Pharm 2001;219: 95–105. 20. Watkinson RM, Guy RH, Hadgraft J, Lane ME. Optimisation of cosolvent concentration for topical drug delivery – II: influence of propylene glycol on ibuprofen permeation. Skin Pharmacol Physiol 2009;22:225–230.
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21. Watkinson RM, Herkenne C, Guy RH, et al. Influence of ethanol on the solubility, ionization and permeation characteristics of ibuprofen in silicone and human skin. Skin Pharmacol Physiol 2009;22:15–21. 22. Bhatia KS, Gao S, Singh J. Effect of penetration enhancers and iontophoresis on the FT-IR spectroscopy and LHRH permeability through porcine skin. J Control Release 1997;47:81–89. 23. Tseng RL, Wu FC, Juang RS. Characteristics and applications of the Lagergren’s first-order equation for adsorption kinetics. J Taiwan Inst Chem Eng 2010;41:661–669. 24. Anissimov YG, Roberts MS. Diffusion modeling of percutaneous absorption kinetics: 4. Effects of a slow equilibration process within stratum corneum on absorption and desorption kinetics. J Pharm Sci 2009;98:772–781. 25. Cestari AR, Vieira EFS, Matos JDS, dos Anjos DSC. Determination of kinetic parameters of Cu(II) interaction with chemically modified thin chitosan membranes. J Colloid Interface Sci 2005; 285:288–295. 26. Mitragotri S, Anissimov YG, Bunge AL, et al. Mathematical model of skin permeability: an overview. Int J Pharm 2011;418:115–129. 27. Hansch C, Leo A, Hoekman D. In exploring QSAR: hydrophobic, electronic and steric constants. Washington, DC: American Chemical Society; 1995. 28. Florence AT, Attwood D. Physicochemical principles of pharmacy. 4th ed. London: Pharmaceutical Press; 2006. 29. Rowe RC, Sheskey PJ, Quinn ME. Handbook of pharmaceutical excipients. 6th ed. Gurnee (IL): Pharmaceutical Press; 2009. 30. Bolzinger M-A, Brianc¸on S, Pelletier J, Chevalier Y. Penetration of drugs through skin, a complex rate-controlling membrane. Curr Opin Colloid In 2012;17:156–165. 31. Shen L-N, Zhang Y-T, Wang Q, et al. Enhanced in vitro and in vivo skin deposition of apigenin delivered using ethosomes. Int J Pharm 2014;460:280–288. 32. Yang Z, Teng Y, Wang H, Hou H. Enhancement of skin permeation of bufalin by limonene via reservoir type transdermal patch: formulation design and biopharmaceutical evaluation. Int J Pharm 2013;447:231–240. 33. Valia KH, Chien YW, Shinal EC. Long-term skin permeation kinetics of estradiol (I): effect of drug solubilizer-polyethylene glycol 400. Drug Dev Ind Pharm 1984;10:951–981. 34. Addicks WJ, Flynn GL, Weiner ND, Curl RL. Drug delivery from topical formulations: theoretical prediction and experimental assessment. In: Osborne DW, Amann AH, eds. Topical drug delivery formulations. New York: Marcel Dekker; 1990:221–244. 35. Bommannan D, Potts RO, Guy RH. Examination of the effect of ethanol on human stratrum corneum in vivo using infrared spectroscopy. J Control Release 1991;16:299–304. 36. Pershing LK, Silver BS, Krueger GG, et al. Feasibility of measuring the bioavailability of topical betamethaosne dipropionate in commercial formulations using drug content in skin and skin blanching assay. Pharm Res 1992;9:45–51. 37. Ribaud C, Garson JC, Doucet J, Le´veˆque JL. Organization of stratum corneum lipids in relation to permeability: influence of sodium lauryl sulfate and preheating. Pharm Res 1994;11: 1414–1418. 38. Van der Merwe D, Riviere JE. Effect of vehicles and sodium lauryl sulphate on xenobiotic permeability and stratum corneum partitioning in porcine skin. Toxicology 2005;206:325–335. 39. Me´ndez-Dı´az JD, Prados-Joya G, Rivera-Utrilla J, et al. Kinetic study of the adsorption of nitroimidazole antibiotics on activated carbons in aqueous phase. J Colloid Interface Sci 2010;345: 481–490. 40. Lee CK, Uchida T, Kazuhisa K, et al. Skin permeability of various drugs with different lipophilicity. J Pharm Sci 1994;83:562–565. 41. Gibbs S, Vietsch H, Meier U, Ponec M. Effect of skin barrier competence on SLS and water-induced IL-1a expression. Exp Dermatol 2002;11:217–223. 42. Lee CH, Maibach HI. Sodium lauryl sulfate. In: Chew A-L, Maibach HI, eds. Irritant dermatitis. Berlin: Springer-Verlag; 2006:257–267.
RESEARCH ARTICLE
Skin deposition and permeation of finasteride in vitro: effects of propylene glycol, ethanol and sodium lauryl sulfate Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Nyu Medical Center on 04/16/15 For personal use only.
Ekapol Limpongsa1, Napaphak Jaipakdee1,2, and Thaned Pongjanyakul1 1
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand and 2Center for Research and Development of Herbal Health Products, Khon Kaen University, Khon Kaen, Thailand Abstract
Keywords
The objective of this study was to investigate the effects of propylene glycol (PG), ethanol (EtOH) and sodium lauryl sulfate (SLS) on the in vitro deposition and permeation of finasteride (FNS). A side-by-side diffusion cell mounted with a pig ear skin and a saturated solution of FNS in PG (10, 20% v/v), EtOH (10, 20% v/v) or SLS (0.5, 1% w/v) vehicles were used. Incorporation of PG, EtOH or SLS caused a significant increase in FNS solubility both in the solution and on the skin with SLS4EtOH4PG. The results obtained from skin deposition studies showed that the FNS deposition rate and time increased in the same order as that of the solubility. The deposition kinetics of FNS solubilized in PG, EtOH and SLS vehicles followed either zero-order, square-root-of-time or pseudo-first-order kinetic models depending on the type and concentration of the enhancer. The permeation studies demonstrated that FNS permeation fluxes were enhanced only by EtOH vehicles. These results suggest that PG and SLS could be used as deposition enhancers, while EtOH could be the effective permeation enhancer of FNS. The obtained results can be used as the considerable insights for formulating the topical and transdermal products of FNS.
Enhancer, finasteride, permeation, skin deposition, solution
Introduction Finasteride (FNS) is an inhibitor of the human 5a-reductase (5a-R) enzyme, which inhibits the conversion of testosterone to dihydrotestosterone (DHT)1,2. There are two types of 5a-R enzymes present in human tissues, referred to as Type 1 and Type 2. In the skin of the scalp, Type 1 5a-R is located in the sebaceous glands, whereas Type 2 5a-R is located in the connective tissue sheaths and dermal papillae of the hair follicles. It has been postulated that the activity of Type 2 5a-R is the cause of androgenetic alopecia (AGA) or male pattern baldness. FNS inhibits Type 2 5a-R more effectively than Type 1 5a-R. Oral FNS was approved by the US FDA in 1997 for the treatment of AGA in men only1. Upon long-term oral administration of FNS, the levels of DHT found in bald scalps were decreased3. FNS is well tolerated and has a favorable adverse event history. The most common adverse events included decreased libido, sexual dysfunction and decreased ejaculate volume resulting in the discontinuation of FNS therapy1. Many studies have reported that the therapeutic effect of FNS solutions and gels included decreased hair loss in both men and women4, regression of sebaceous glandular and ductal hyperplasia in fuzzy rats5 and the re-enlargement of hair follicles in human scalp skin grafts transplanted onto SCID mice2. These studies
Address for correspondence: Ekapol Limpongsa, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Tel: +66 43 362092. Fax: +66 43 362092. E-mail: ekapol@ hotmail.com
History Received 4 July 2014 Revised 3 August 2014 Accepted 5 August 2014 Published online 27 August 2014
encourage the use of FNS via topical application. Moreover, it is more favorable to use a percutaneous approach for the delivery of therapeutic agents directly onto the targeted tissues to reduce first-pass effects and systemic side effects6. Drug transport of a formulation to the systemic circulation is a multistep process which involves (a) dissolution within the formulation and the drug’s subsequent release, (b) partitioning of the drug into the stratum corneum (SC) of the skin, (c) diffusion through the SC, (d) partitioning of the drug from the SC into the aqueous viable epidermis, (e) diffusion through the viable epidermis and into the upper dermis and (f) uptake into the local capillary network and eventually into the systemic circulation7. Human skin is an effective barrier to chemical permeation8. Many strategies have been suggested to overcome the low permeability of drugs through the skin. A popular approach is the use of permeation enhancers that interact with skin constituents to promote drug flux9. An effective permeation enhancer may increase the diffusivity of the skin, partitioning of the drug between the formulation and the skin or the effective concentration of the drug in the vehicle8. To administer FNS via a topical route, a preparation that improves skin deposition (penetration into skin) while reducing skin permeation (systemic absorption) is desirable10. Javadzadeh et al.11 reported that the permeation of FNS through rat skin decreased when cetyltrimethylammonium bromide or sodium lauryl sulfate (SLS) was used; unfortunately deposition was not measured. Many FNS vesicles including liposomes, niosomes, ethosomes and polymersomes have been previously studied6,10,12,13. FNS permeation flux values were 1.23-fold lower
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for liposomes10,13, 2.31-fold higher for ethosomes6 and 1.33- to 1.57-fold higher for polymersomes12 when compared to hydroethanolic solutions of FNS (30%). The hydroethanolic solution allowed for higher FNS skin deposition than liposomes; however, the deposition levels were lower than those obtained with ethosomes. In another study, liposomes enhanced FNS skin deposition; however, problems related to chemical and microbial stability were encountered and thus limit their applications14. The objective of this study was to investigate the effects of propylene glycol (PG), ethanol (EtOH) and SLS on skin deposition and permeation of FNS in vitro. The in vitro skin deposition kinetics were also studied. Skin deposition was defined as the amount or concentration of drug deposited in the skin15. PG and EtOH are inexpensive, nontoxic, and well-tolerated co-solvents, whereas SLS is an anionic surfactant that can penetrate and interact strongly with the skin. Their efficacy and action have been reported in the literature16–21. The results obtained from this study can be used as the insight information for formulating the topical solution of FNS.
(PBS) at 32 C for 15 min and was then measured the thickness using a dial thickness gauge (PeacockÕ , Labtek, Scotts Valley, CA). The hydrated skin was mounted between the donor and receptor compartments with a clamp and was further hydrated with PBS for 45 min. The saturated solution of FNS (3 mL) was added to the donor compartment, which was in contact with the SC side of the skin. The receptor compartment was filled with 3 mL of 40% v/v PEG400 in deionized water. After 0.5, 1, 2, 3, 4 or 10 h, the skin samples were removed and washed on both sides with 0.5 mL of water (3 times) followed by 0.5 mL of methanol (3 times). The part of each skin that directly contacted the saturated solution of FNS (area of 0.694 cm2) was separated using scissors and dried at 50 C for 24 h. The dried skin was cut into small pieces, and FNS was extracted with methanol. The extraction was performed twice. The samples were analyzed for FNS content by HPLC. The deposition amount of FNS per unit area (Qs; mg/cm2) was plotted against time (h).
Materials and methods
The saturated deposition amount per unit area (Qs,sat; mg/cm2) was considered the average deposition amount of the plateau portion of the deposition profile. The volume of skin was calculated by multiply the diffusion area with the skin thickness. The deposition concentration was the deposition amount per volume of skin (Cs; mg/mL). The saturated deposition concentration in the skin or the skin solubility (Cs,sat) was the average deposition concentration of the plateau portion on the deposition profile. The deposition time to saturation (Tdep; h) was the time Qs,sat obtained from the deposition profile. The partition coefficient of FNS from the vehicle onto the skin (K) was obtained using the saturated concentration in the vehicle (Cv,sat or solubility in vehicle) and the saturated concentration in the skin (Cs,sat or solubility in skin) as shown in Equation (1)7.
Materials Finasteride (FNS) was purchased from Aurisco Pharmaceutical, Shanghai, China. Propylene glycol (PG) and sodium lauryl sulfate (SLS) were obtained from S. Tong Chemicals, Bangkok, Thailand. Ethanol (EtOH) and methanol were purchased from QReC, New Zealand. Polyethylene glycol 400 (PEG400) was purchased from Merck KGaA, Darmstadt, Germany. Deionized water was used throughout the studies. All chemicals were of reagent or high-performance liquid chromatography (HPLC) grade. Methods
Data analysis
Solubility study Excess amounts of FNS were added to each solvent. The mixture was sonicated for 1 h and then equilibrated at 32.0 ± 0.5 C in a shaking water bath (Digital Temperature Controller, Polyscience Inc, Warrington, PA) for 72 h. Mixtures were then filtrated through a membrane filter (0.45 mm, 13 mm, Millipore filter, Millipore, Billerica, MA). The filtered solution was then diluted and analyzed for FNS content by HPLC assay at 210 nm as described below. Skin preparation Porcine ears were obtained from a local slaughter house and cleaned with water. After soaking the ears in water at 60 C for 45 s, the intact epidermis was peeled off with forceps, washed with water and kept at 20 C until use (within 4 weeks)22. The frozen skin was thawed at an ambient temperature before use.
K ¼
ð1Þ
Kinetics of deposition The saturated solutions were used in this study to ensure that the drug is at its maximum thermodynamic activity. During the deposition process, it is thought that the rate is proportional to the difference between the amount of deposition at time t and the deposition capacity or saturated deposition amount of drug in the skin (Qs,sat Qs)23. The zero-order, square-root-of-time and pseudo-first-order kinetic models were used to interpret the experimental kinetic data and are represented below by Equations (2)–(4) [or 5], respectively23–25,
In vitro skin deposition study Saturated solution preparation. Saturated solutions of FNS were prepared by sonicating the mixture of excess FNS and solvent (1 h) and then equilibrating at 32.0 ± 0.5 C (72 h). This mixture was used for the deposition and permeation studies without any further filtration. The in vitro deposition of FNS from the saturated solution into the pig ear skin was conducted using a side-by-side diffusion cell with a diffusion area of 0.694 cm2 (Crown Glass Company, Somerville, NJ). The system was connected to a water bath maintained at a temperature of 32.0 ± 0.5 C. A thawed skin was hydrated by immersing in a pH 7.4 phosphate buffered saline
Cs, sat Cv, sat
or
Qs, sat Qs ¼ k0 t
ð2Þ
Qs, sat Qs ¼ ksq t1=2
ð3Þ
Qs, sat Qs ¼ Qs, sat expk1 t
ð4Þ
lnðQs, sat Qs Þ ¼ lnðQs, sat Þ k1 t
ð5Þ
where Qs is the deposition amount of the drug at time t, Qs,sat is the saturated amount of drug in the skin and k0, ksq and k1 are the deposition rate constants of the zero-order, square-root-of-time and pseudo-first-order kinetic models, respectively.
Skin deposition and permeation of finasteride in vitro
DOI: 10.3109/10837450.2014.954727
The deposition rate constants (k0, ksq and k1), the determination coefficient (R2) and the calculated saturated amount of drug in the skin (Qs,sat,cal) were obtained by fitting the kinetic models to the experimental data. The experimental data were compared with the predicted curves using zero-order, square-root-of-time and pseudofirst-order models by using the normalized standard deviation [Equation (6)]23 as defined by sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 P ðQs, exp Qs, cal Þ=Qs, exp ð6Þ Dqð%Þ ¼ 100 N 1
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where the subscripts exp and cal denote the experimental and calculated values, respectively, and N is the number of data points. In vitro skin permeation study The in vitro permeation of FNS from the saturated solution through the pig ear skin was conducted using a side-by-side diffusion cell under conditions similar to those used in the skin deposition studies. At predetermined times, 1.0-mL samples were taken from the receptor compartment and equal volumes of 40% PEG400 in deionized water were immediately added after each sampling. The concentration of FNS was analyzed by HPLC. The cumulative amount of drug that permeated the skin was plotted against time. Data analysis The steady-state flux (Jss), the permeability coefficient of the skin (Ps) and the diffusivity of the skin (Ds) are defined by Equations (7)–(9)26. Jss ¼ Ps Cv, sat ¼
K Ds Ds Cs, sat Cv, sat ¼ L L
ð7Þ
3
consisted of methanol: water at a volume ratio of 75:25. The retention time of FNS was approximately 7 min. The standard curve was linear over a concentration range of 0.8–10 mg/mL with an R2 value 4 0.99. The day-to-day relative standard deviations (RSD) for this assay were less than 5%. Statistical analysis Each experiment was repeated at least three times. The results are expressed as the mean ± S.D. One-way analysis of variance was used to test the statistical significance of differences among groups. Statistical significance of the differences of the means was determined by Student’s t-test. All statistical tests were run using the SPSS program for MS Windows, release 19 [SPSS (Thailand) Co. Ltd., Bangkok, Thailand]. The significance was determined with 95% confident limits ( ¼ 0.5) and was considered significant at a level of p less than 0.05.
Results FNS solubility in the vehicle The effects of PG (10 and 20%), EtOH (10 and 20%) and SLS (0.5 and 1.0%) on FNS solubility (at 32 C) were determined and are reported in Table 1. The solubility of FNS in water was 42.3 ± 1.9 mg/mL. Addition of co-solvents (PG or EtOH) significantly improved FNS solubility. These solubility improvements were significantly pronounced at higher concentrations of co-solvent (p50.05). The solubility of FNS in different solvents occurred in the following order: 20% EtOH420% PG410% EtOH410% PG, respectively. Addition of SLS could also increase the solubility of FNS; the solubility was dramatically increased approximately 32- and 68-fold when 0.5 or 1.0% SLS was used, respectively. FNS skin deposition
Ps ¼
K Ds L
ð8Þ
Ds ¼
Jss L Cs, sat
ð9Þ
where L is the thickness of skin. HPLC analysis FNS content was determined by an HPLC system (Perkin-Elmer, Waltham, MA) consisting of a UV detector (model 1022 LC plus) and a pump (series 200 LC). The chromatographic separation was achieved on a Hypersil Gold C-18 column (250 4.6 mm, 5 mm; Thermo Electron Corporation, Waltham, MA) with a flow rate of 1 mL/min and UV detection at 210 nm. The mobile phase
The amount of FNS deposited in skin (Qs) was plotted against time as shown in Figure 1. It could be seen that the FNS skin deposition profiles obtained from the PG, EtOH and SLS solutions were higher than that of the control solution. SLS resulted in the highest FNS deposition as compared to those obtained from EtOH and PG, respectively. The increase in PG, EtOH or SLS concentrations in the vehicle resulted in higher skin deposition levels. Table 1 reports the skin solubility (Cs,sat), partition coefficient (K), saturated deposition amount (Qs,sat) and deposition time (Tdep) of FNS in various vehicles. The addition of PG, EtOH or SLS resulted in an increase in skin solubility and saturated skin deposition levels. The increase in PG, EtOH or SLS concentration resulted in a significant increase in solubility and deposition amount of FNS in skin (p50.05). The ranked order of FNS skin solubility and deposition amounts are the same as the vehicle solubility (Cv,sat) rank.
Table 1. Effects of propylene glycol (PG), ethanol (EtOH) and sodium lauryl sulfate (SLS) on the vehicle solubility (Cv,sat), the skin solubility (Cs,sat), the partition coefficient (K), the saturated deposition amount (Qs,sat) and the deposition time to saturate (Tdep) of finasteride from saturated solution in pig ear skin at 32 C (mean ± SD, n ¼ 3). Vehicles
Cv,sat (mg/mL)
Cs,sat (mg/mL)
K
Qs,sat (mg/cm2)
Tdep (h)
Water (control) 10% PG 20% PG 10% EtOH 20% EtOH 0.5% SLS 1.0% SLS
42.3 ± 1.9 89.9 ± 5.2a 202.5 ± 4.1a 124.2 ± 6.5a 362.4 ± 8.8a 1360.3 ± 43.2a 2869.4 ± 83.2a
61.0 ± 6.0 104.9 ± 14.3a 177.1 ± 32.0a 148.6 ± 11.8a 335.8 ± 53.9a 718.3 ± 57.5a 1237.6 ± 67.6a
1.5 ± 0.1 1.2 ± 0.2a 0.9 ± 0.2a 1.2 ± 0.1a 0.9 ± 0.2a 0.5 ± 0.0a 0.4 ± 0.0a
9.5 ± 0.1 16.7 ± 0.8a 26.6 ± 1.1a 21.9 ± 1.3a 59.1 ± 3.0a 114.8 ± 1.6a 209.3 ± 1.0a
1.9 3.4 3.4 3.8 4.0 3.0 3.0
a
Significant different from control at p50.05.
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The partition coefficient can be calculated based on vehicle and skin solubility according to Equation (1). Surprisingly, the addition of PG (10%), EtOH (10%) or SLS (0.5%) led to a significant decrease in the partition coefficient when compared to the control. Increasing the PG or SLS concentration had no further effect on the partition coefficient. However, the partition coefficient of FNS significantly decreased (p50.05) as EtOH concentration increased (10–20%). The deposition time to saturation (Tdep) was the time that reached the saturated deposition amount per unit area, obtained from the deposition profile. It should be noted that there was no lag time for deposition of all vehicles used in this study. As can be seen in Table 1, the deposition times of FNS in all vehicles containing permeation enhancers were longer than those of the control and ranged from 3.0 to 4.0 h. EtOH vehicles yielded the longest deposition time as compared to those obtained from the others.
and the normalized standard deviation (Dq) for all vehicles. The examples of the deposition kinetics of FNS in water, 20% PG and 0.5% SLS solutions obtained from different kinetic models are depicted in Figure 2. It is obvious from the rate constants that the deposition process was accelerated by the incorporation of PG, EtOH or SLS into the vehicle. This indicates that the deposition of FNS from the vehicles containing these enhancers onto the skin was more rapid and favorable than water alone. The effect on the rate of deposition was more pronounced when higher concentrations of PG, EtOH or SLS were used. Considering that the best-fit model shall yield the high R2 together with the low Dq values, the FNS deposition process from water, 10% PG and 20% EtOH vehicles followed the squareroot-of-time model (Table 2). In contrast, the FNS deposition process in 20% PG and 10% EtOH vehicles followed the pseudofirst-order model, whereas in the vehicle containing SLS followed the zero-order model.
Deposition kinetics
FNS skin permeation
The kinetic provides valuable insights of the deposition mechanism involved. In this study, the zero-order, square-root-of-time and pseudo-first-order kinetic models [Equations (2), (3) and (5), respectively] were selected to fit the deposition kinetic data. Table 2 shows the values of the rate constants obtained from each kinetic model (k0, ksq and k1), the determination coefficient (R2), the calculated saturated deposition amount of FNS, Qs,sat,cal
In vitro permeation of saturated FNS solutions through the pig ear skin was studied and the permeation-time profiles are shown in Figure 3. The permeation profiles of FNS in PG solutions (10 and 20%) and SLS solutions (0.5 and 1.0%) were comparable to those of the control vehicle (water), while profiles from EtOH solutions (10 and 20%) were higher than those of the control. Table 3 shows the corresponding steady-state flux (Jss), permeability coefficients
Figure 1. Effects of vehicle types (Water, PG, EtOH and SLS) on skin deposition amount (Qs) versus time plots of FNS from saturated solution (n ¼ 4).
Table 2. Deposition rate constants (k0, ksq and k1) and saturated deposition amount (Qs,sat) of finasteride from various kinetic models. Zero-order Vehicles Water (control) 10% PG 20% PG 10% EtOH 20% EtOH 0.5% SLS 1.0% SLS
Square root of time
Pseudo-first-order
k0 (mg/cm2/h)
R2
Qs,sat,cal (mg/cm2)
Dq (%)
ksq (mg/cm2/h1/2)
R2
Qs,sat,cal (mg/cm2)
Dq (%)
k1 (h1)
R2
Qs,sat,cal (mg/cm2)
Dq (%)
3.94 5.74 7.95 9.07 19.70 42.15 70.48
0.856 0.972 0.964 0.897 0.839 0.998 0.997
8.1 15.8 25.1 19.1 51.1 113.1 206.1
36.0 19.5 21.8 31.1 37.1 6.8 8.4
6.06 8.25 11.52 13.72 30.69 58.33 97.47
0.985 0.978 0.985 0.999 0.990 0.930 0.928
9.4 17.2 27.2 21.8 57.8 121.7 220.5
10.9 15.8 16.6 4.6 13.3 34.6 32.1
1.05 1.08 0.48 1.13 0.62 0.70 0.59
0.986 0.939 0.997 0.998 0.955 0.978 0.980
8.8 14.7 29.4 22.1 52.2 124.6 221.2
17.1 24.6 11.1 4.3 31.9 29.8 21.8
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DOI: 10.3109/10837450.2014.954727
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Figure 2. Deposition kinetics of FNS from (a) water, (b) 20% PG and (c) 0.5% SLS solution in pig ear skin.
(Ps), skin diffusivities (Ds) and lag times (Tlag) of FNS permeations. It can be seen that PG and SLS had an insignificant effect on the flux. In contrast, EtOH resulted in a significantly higher flux when compared to the control. Increase in EtOH concentration from 10% to 20% resulted in significant increases of the FNS flux (p50.05). The permeability coefficient of FNS through the pig ear skin decreased significantly when PG, EtOH or SLS was added to the chamber. The increasing PG, EtOH or SLS concentrations in the vehicle led to significant decreases in the permeability coefficients (p50.05). PG, EtOH or SLS had insignificant effect on the lag time when compared to the control. However the skin diffusivity which was calculated using Equation (9) decreased significantly when the
vehicle contained PG, EtOH or SLS. Interestingly, increase the EtOH concentration from 10% to 20% significantly increased the skin diffusivity.
Discussion FNS is a lipophilic drug with low solubility in water and a high log Poct value27. The improvement of FNS solubility by PG and EtOH was attributed to their co-solvent effects on the polarity of the vehicle. EtOH resulted in higher solubility improvements when compared to those obtained from PG. This effect could be attributed to the lower polarity of EtOH (solubility parameter, , of 12.74 cal1/2 cm3/2) when compared to PG ( of 14.76 cal1/2 cm3/2)28. Nevertheless, the highest solubility increase was
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Figure 3. Effects of vehicle types (a: PG; b: EtOH; c: SLS) on cumulative permeation-time profiles of FNS from saturated solution (n ¼ 4).
Table 3. Effects of propylene glycol (PG), ethanol (EtOH) and sodium lauryl sulfate (SLS) on the steady-state flux (Jss), the permeability coefficient (Ps), the skin diffusivity (Ds) and the lag time (Tlag) of finasteride from saturated solution across pig ear skin at 32 C (mean ± SD, n ¼ 4).
Vehicles Water (control) 10% PG 20% PG 10% EtOH 20% EtOH 0.5% SLS 1.0% SLS a
Ps Jss (mg/cm2/h) (10 3 cm/h) 2.8 ± 0.6 2.8 ± 0.5 3.2 ± 0.3 3.9 ± 0.5a 8.0 ± 0.8a 2.0 ± 0.1 2.4 ± 0.3
67.0 ± 13.5 31.3 ± 6.0a 15.9 ± 1.7a 31.7 ± 4.3a 22.1 ± 2.3a 1.5 ± 0.1a 0.8 ± 0.1a
Significantly different from control at p50.05.
Ds (103 cm2/h)
Tlag (h)
7.1 ± 1.1 4.3 ± 0.9a 2.9 ± 0.3a 3.7 ± 0.7a 4.9 ± 0.2a 0.4 ± 0.0a 0.3 ± 0.1a
1.7 ± 0.5 2.3 ± 0.5 2.4 ± 0.2 2.1 ± 0.1 2.2 ± 0.5 2.0 ± 0.5 2.6 ± 0.5
observed for the SLS solution, which could be due to the effects of micellar solubilization. The critical micelle concentration (CMC) of SLS is only 0.24%29. In vitro skin deposition and permeation of saturated FNS solutions was studied using a side-by-side diffusion cell. A 40% PEG400 solution was used as a receiver medium in order to maintain the sink condition throughout the experiment30. PEG400 is one of the few solvents widely used to increase the solubility of the investigated drug in the receiver fluid. Depends on the hydrophobicity of the investigated compounds, various concentrations of solvent(s) were used22,31,32. In the case of FNS, it was found that the solubility of FNS in 40% PEG400 solution was sufficient to obtain the sink condition. The solubility of FNS at 32 C in 40% PEG400 solution (786.6 ± 5.3 mg/mL) was 19-fold greater than it was in PBS (540 mg/mL). It has been
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DOI: 10.3109/10837450.2014.954727
demonstrated that the incorporation of up to 40% v/v PEG400 does not influence the barrier properties of SC33. The transdermal/topical delivery is a complex phenomenon governing with the release from the vehicle, the enhancing potency of vehicle and partitioning of drug into the skin. The release of drug from a vehicle into the skin and its diffusion across the skin are controlled by physicochemical factors sensitive to the molecular properties of the drug, the vehicle and the skin34. The delivery process can be affected by interactions that occur between the drug and skin, vehicle and skin, drug and vehicle, and drug, vehicle and skin. Increasing a drug’s solubility in the vehicle leads to larger concentration gradient between the vehicle and the skin. This idea may be responsible for the increases observed in the deposition rate of FNS. PG and EtOH may deposit onto the skin17,21,35,36 whereas SLS may disrupt the SC layer of the skin37,38, resulting in a decrease in the barrier property of the skin and subsequently enhance FNS solubility and deposition rate. The higher affinity of drug to the vehicle could be the reason for the decreases in FNS partition coefficient. Addition of PG or EtOH to water rendered the vehicle less polar. The affinity of FNS dissolved in the vehicle increased with increasing ratios of PG or EtOH. As a result, the partition coefficient decreased. The relationship between the solubility parameters (indicating polarity) and the partition coefficient with a high coefficient of determination is shown in Figure 4. Some of the PG or EtOH from the vehicle could also partition, diffuse and deposit on the skin17,19. EtOH has a lower molecular weight but a greater log Poct value than PG27. Therefore, EtOH has a higher affinity for the skin than PG. The higher amounts of EtOH may be allocated for deposition onto the skin when compared to PG. This phenomenon might cause the longer deposition times of FNS in the EtOH vehicle when compared to that in the PG vehicle. The skin deposition process of FNS from 10% PG and 20% EtOH vehicles followed the square-root-of-time model, indicating the limitation of FNS diffusion within the skin7,25. In contrast, the FNS deposition process in 20% PG and 10% EtOH vehicles followed the pseudo-first-order model, indicating that the transport may be controlled by a chemical reaction between FNS and the active sites of the SC in skin39. PG in the vehicle may extract some water from the SC19, leading to an increase in the SC barrier and a decrease in FNS diffusivity in the skin. At low concentrations, EtOH could increase the fluidity of
Skin deposition and permeation of finasteride in vitro
7
the lipoidal pathway in the SC19 and enhance FNS skin affinity. Therefore, the diffusivity in skin was decreased. As can be seen from Equation (8), the decrease of the diffusivity and partition coefficient in skin resulted in the decrease of the permeability coefficient of FNS from PG and EtOH vehicles. From Equation (7), permeation flux is a product of permeability coefficient and concentration of drug in the apply vehicle. In the case of PG vehicles, the slight increase in FNS solubility (concentration) in the vehicle might compensate the decrease in permeability coefficient; therefore, the FNS fluxes from the PG vehicles were not different from that of the control. However, enhancement of FNS flux from the EtOH vehicles may be the result of higher concentrations of FNS in the vehicles when compared to those from the PG vehicles and the control. Incorporation of SLS at concentrations above CMC caused the formation of micelles around FNS and the enhancement of FNS solubility in the vehicles. SLS can penetrate and strongly interact with the skin leading to the enhancement of the amount, solubility and time of FNS deposition in the skin16,40. However, FNS may have a high affinity for micelles; thus, the fraction of unassociated diffusing species was lower24. The partition coefficient of FNS was also very low. Because hydrophobic interactions of the SLS alkyl chain with the skin structure leaves an end sulfate group of the surfactant exposed, it creates additional sites in the membrane that can permit an increase in skin hydration37. The FNS skin deposition process from the vehicle containing SLS followed the zero-order model, indicating an increase in the number of deposition sites. The diffusion of FNS into the skin was dependent on the diffusion of micelles. SLS micelles exhibit sizes greater than FNS and can strongly interact with the skin; therefore, their diffusion was very slow. The skin diffusivity of 0.5% and 1.0% SLS solutions were approximately 18 and 24 times less than that of the control. The very low diffusivity and partition coefficient in skin caused the very low permeability coefficient of FNS from SLS vehicles. As compared to the control, the permeability coefficient of FNS from 1.0% SLS solution decreased for 84 times. Nevertheless, FNS fluxes from the vehicles containing SLS were not significantly different from that of the control. This is attributed to the effect of SLS on the FNS solubility in the vehicle. SLS is an anionic surfactant that is found in many therapeutic preparations9. It has been shown that SLS application disrupted the skin barrier function resulting in increased transdermal water loss41,42. The effect of SLS on excised human skin was previously studied and found that SLS at concentration up to 5% had no visible effects on tissue architecture when compared with untreated samples41. The report of Nokhodchi et al.18 which performed permeation experiments with rat skin of lorazepam as a function of SLS concentrations (0.5, 1.0, 2.5, 5.0% w/w) suggested that the extensive skin damage might occur when the concentration of SLS higher than 2.5%.
Conclusions
Figure 4. Relationship between solubility parameter and partition coefficient of FNS from water, PG and EtOH solutions (Y ¼ 0.3257x 6.1718, R2 ¼ 0.9934).
The effects of PG, EtOH and SLS on FNS skin deposition and permeation were studied. SLS (0.5 and 1%) yielded the highest solubility of FNS in the vehicles, and therefore skin deposition. In contrast, permeation studies demonstrated that only the EtOH containing vehicle (10 and 20%) could enhance FNS permeation fluxes. The results of this study suggest that EtOH could be the effective permeation enhancer; whereas, when the local effects are desirable, PG and SLS could be used as deposition enhancers of FNS. These results provided the considerable insights for selecting a suitable enhancer for transdermal or topical products of FNS. Nevertheless, further investigations regarding skin irritation effect of these vehicles shall be performed.
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Declaration of interest The authors wish to thank the Thailand Research Fund (MRG 5380119), the Commission of Higher Education and Khon Kaen University, Ministry of Education, Thailand, for financial support. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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