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Topical delivery of retinyl ascorbate co-drug
International Journal of Pharmaceutics 280 (2004) 113–124
Topical delivery of retinyl ascorbate co-drug 1. Synthesis, penetration into and permeation across human skin Kasem Abdulmajed, Charles M. Heard∗ Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK Received 21 October 2003; received in revised form 15 April 2004; accepted 10 May 2004
Abstract A novel synthetic technique was used to synthesise the co-drug retinyl ascorbate (RA-AsA) ester from all-trans-retinyl chloride (RA) and l-ascorbic acid (AsA) suspended in ethanol at low temperature. Its log P, solubility in a Me:PBS, 50/50 at pH 4.8 and degradation constant were determined. The flux and permeation coefficient were determined using heat separated human skin membrane, and skin penetration was determined by tape stripping using full thickness human. All experiments were performed in parallel with retinyl palmitate (Rol-Pal) and ascorbyl palmitate (AsA-Pal), which are used in commercial topical formulations. RA-AsA exhibited favourable log P (2.2), with stability much greater than RA and AsA, but similar stability to Rol-Pal and AsA-Pal. The flux of RA-AsA was lower than for Rol-Pal and AsA-Pal. RA-AsA also demonstrated higher skin retention than the other two esters, but delivered more RA and AsA to the viable epidermis than retinol from Rol-Pal and ascorbic acid from AsA-Pal. Overall, the data suggest the potential value of RA-AsA co-drug for the purpose of treating damage to skin resulting from UV-induced production of free radicals. © 2004 Elsevier B.V. All rights reserved. Keywords: Co-drug; Retinoic acid; Retinyl ascorbate; Anti-oxidant; Skin permeation; Tape stripping; Ascorbic acid
1. Introduction Epidermal keratinocytes are exposed to external and internal oxidative stress. Ultraviolet irradiation induces the generation of reactive oxygen species (ROS) and the lipid peroxidation of the plasma membrane (Krutmann et al., 1992). Physiological levels of ultraviolet B induce intracellular hydrogen peroxide (H2 O2 ) generation in human epidermal keratinocytes (Peus et al., 1988). Infiltrating polymorphonuclear leukocytes and macrophages also generate ROS including superoxide anion radical, H2 O2 and hydroxyl ∗ Corresponding author. Tel.: +44 29 20875819; fax: +44 29 20875819. E-mail address: [email protected] (C.M. Heard).
radical (Baggiolini et al., 1993; Ishii et al., 1999). ROS are implicated in ultraviolet UV-light induced damage to skin (Perchellet and Perchellet, 1989) which could also be an initiator in the pathogenesis of photoaging and skin cancer (Dalle Carbonare and Pathak, 1992; Emerit, 1992; Shindo et al., 1994; Nachbar and Korting, 1995). Antioxidants that protect the skin against ROS include various low molecular weight antioxidants (glutathione, tocopherol and ubiquinol) and antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and thioredoxin reductase) (Fuchs et al., 1989, Pence and Naylor, 1990; Shindo et al., 1993; Nachbar and Korting, 1995). Topical administration of antioxidants provides an efficient way to enrich the endogenous cutaneous protection system and thus may be a successful strategy
0378-5173/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2004.05.008
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for diminishing ultraviolet radiation-mediated oxidative damage in the skin (Khettab et al., 1988; Weber et al., 1997). Retinoic acid (RA) is highly lipophilic (log P = 4.6) and the most potent form of the retinoids, which exert a wide variety of effects in the regulation of cell division, and possess some antioxidant properties (Khettab et al., 1988; Boreman and Napoli, 1996). l-Ascorbic acid (AsA) is a strong and powerful water-soluble antioxidant that efficiently protects important organic and biological molecules against ox-
idative degradation by inhibiting free radical initiated lipid peroxidation, a process believed to be responsible for photoaging and the induction of collagen synthesis (Pinnell, 1987; Philips et al., 1994). It functions even better in conjunction with other antioxidants, such as Vitamin E (tocopherols) and carotenoids, by establishing a peculiar recycling system with a synergic effect (Darr et al., 1996). However, due to the very low solubility of AsA in non-aqueous media (log P = −3.4), it is not well-suited to dermal application. In addition,
Fig. 1. Chemical structures of the test compounds.
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both RA and AsA are highly unstable when exposed to light, air and elevated temperatures. RA contains a carboxylic acid group, whereas AsA contains hydroxyl functionality, thus allowing the formation of an ester co-drug. Co-drugs were first described by Jones (1990), although the first report of a co-drug for topical application was by Nazemi et al. (2000). It is hypothesised that the co-drug retinyl ascorbate (RA-AsA) would demonstrate enhanced dermal uptake properties relative to both parent moieties as it would be more lipophilic (log P = 2.2) and be subject to greater uptake into the lipoidal domains of the stratum corneum (Walter and Kurz, 1988). Furthermore, the ester linkage could confer greater stability to both parent compounds, allowing them to penetrate deeper into the skin and provide combined regulatory/antioxidant properties, once the ester bond is hydrolysed in situ, either enzymatically or chemically. Thus, dermal application should be enhanced for protection against UV-induced oxidative damage to the skin. In this study RA-AsA co-drug was synthesised and its solubility and stability in different vehicles determined, before being studied in vitro for its suitability for application to human skin. For comparative purposes, retinyl palmitate (Rol-Pal) and ascorbyl palmitate (AsA-Pal), both of which are used in topical formulations for cosmetic and medicinal purposes (Boehnlein et al., 1994; Darr et al., 1996) were also examined. The structures of the compounds studied in the work are shown in Fig. 1.
2. Materials and methods 2.1. Materials All-trans-retinoic acid (RA), l-ascorbic acid (AsA), all-trans-retinol (Rol), all-trans-retinol palmitate (Rol-Pal), palmitic acid (all of purity > 99), phosphorous trichloride and OH-substituted anion exchange resin were obtained from Sigma (Poole, UK). Ultrafree-CL 2 ml tubes, containing low-binding Durapore PVDF 0.22 m filter inserts, were from Millipore (Bedford, MA, USA). D-Squame skin sampling discs, 14 mm, were from CuDerm Corporation (Dallas, Texas, USA). All other reagents and solvents were from Fisher (Loughborough, UK). Female
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abdominal human skin was obtained post-cosmetic surgery and frozen immediately. 2.2. Synthesis of retinyl ascorbate Firstly, retinyl chloride was synthesised. A solution of 1 mM phosphorous trichloride in 6 ml of dry benzene was added slowly to a stirring suspension of 1.5 g (1.4 mM) of all-trans-retinoic acid in15 ml of benzene and stirred continuously at 25 ◦ C, for 1 h. The reaction mixture was then allowed to cool in an ice bath and was the mixture decanted to separate the precipitate. The solution was then tested for the presence of chloride by reacting 0.5 ml with an ethanolic solution of silver nitrite and potassium iodide yielding a white precipitate of silver chloride. The reaction was followed by TLC, Rf = 0.2 in (Pet:EtO:CHCl3 ), (1:1:1) (Fulmer Shealy et al., 1988). l-Ascorbic acid (1.7 g) was suspended in 30 ml of ethanol and allowed to stand over 2 g of molecular sieves and allowed to dry overnight under light-shield. The sieves were removed and 2 g of dry anion exchange resin was added to the solution. After 30 min, 10 mg of K2 CO3 were added the reaction and 15 ml of retinyl chloride added, drop wise, and the mixture stirred overnight at 30 ◦ C and under light-shield. The reaction was followed by TLC (silica gel/1:2:1 Pet:EtO:CHCl3 ). After 15 h the solvent was evaporated under vacuum and the concentrate dissolved in 250 ml of CH3 OH:CHCl3 and washed with 5 M brine. Two hundred and fifty milliliters of n-hexane was added and cooled to 4 ◦ C. The lower layer was concentrated and purified three times by column chromatography to obtain 264 mg of product in the form of an off-white powder and with a purity of >98.5%. 2.3. Synthesis of ascorbyl palmitate Firstly, palmityl chloride was synthesised. A solution of 2.3 g (0.84 M) of phosphorous trichloride in 20 ml of dry benzene was added slowly to a stirring suspension of 15 g (1.4 M) of palmitic acid in 50 ml of benzene. Stirring was continued, at 25 ◦ C, for 1 h. The reaction mixture was then allowed to cool and then decanted to separate the precipitate. The solution was then tested for the presence of chloride by reacting 0.5 ml with an ethanolic solution of silver nitrite
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and potassium iodide yielding a white precipitate of silver chloride. Ascorbic acid (10 mg) was added to a solution of 50 ml of palmityl chloride in 150 ml of pyridine. The mixture was then stirred for 40 h at 40 ◦ C, under light-shield, and nitrogen. The reaction was followed by TLC (silica gel; PEt:EtO:MeOH, 1:3:2). The reaction mixture was then concentrated under pressure. Ethyl acetate was added to the oil residue, which immediately gave a white precipitate. After filtering, the residue was washed with hot EtOAc and hot water (60–70 ◦ C) and then dried. This was then recrystallized from EtOH:H2 O (4:1) to give 467 mg of white powder. 2.4. Solubility and stability issues AsA was highly soluble in water but of limited solubility in ethanol, whereas RA-AsA, RA, Rol, Rol-Pal, and AsA-Pal are insoluble in water but of varying solubility amounts in ethanol. However, all the compounds are soluble in methanol, but with limited stability. Furthermore, the RA-AsA, RA and AsA exhibited limited stability in an ethanol/water, and methanol/water co-solvents. It was determined (data not shown) that a co-solvent system of Me:PBS, 50:50, pH 4.8, was the most suitable for all six compounds in terms of solubility and stability for the experiment duration. Also, pH 4.8 is near to the pH of human skin surface of ∼5.5. AsA, RA, Rol and RA-AsA are highly unstable on exposure to light, temperature and atmospheric oxygen (Chikakane and Takahashi, 1995). Therefore, their solubility was determined by the shaking and assaying method, i.e. on the basis of the compounds kinetics rather than their thermodynamics. Employing the kinetic method (shaking then assaying) for the thermodynamic method (shaking until equilibrium) traded the uncertainty of a desired form for the certainty of a potentially undesired form (Wilson, 2001). 2.4.1. Solubility An excess of each compound was placed in Ultrafree-CL tubes containing 1 ml solution of methanol and PBS, pH 4.8. The vials were then placed on a blood tube rotator in an incubation oven set at 37 ◦ C for 1 h under light-shield, the tubes were then centrifuged at 12,000 rpm for 5 min, 100 l samples were, then assayed by HPLC (section 2.10.), the ex-
periments were carried out in triplicates, the remainder was used to determine the calibration curve and the lowest detection limit and for the skin experiments. 2.4.2. Stability A 5 ml saturated solution of each compound in Me/PBS was added to glass vials in six sets of x3. Two sets were maintained at room temperature, 37 and 60 ◦ C whilst stirring. One set was shielded from light, the other not. Samples (100 l) were collected from each vial at regular intervals over a period of 56 h. Experiments were terminated at 1 h (for the 60 ◦ C for both shielded and unshielded), 1 h (for the 37 ◦ C unshielded), and 2 h (for those placed at room temperature, unshielded). The samples were then analysed by HPLC. Zero order decomposition rates, k (mg ml−1 h−1 ), were calculated from the equation: A0 − A = kt, where A0 is the initial concentration, A is the remaining concentration at time t, and k is the gradient of [A0 − A] against t. 2.5. log Po/w determination Samples (∼60 mg) were placed in octanol:deionised water (50:150 cm3 ) in duplicates and shaken vigourously on a mechanical shaker, for 30 min. The layers were allowed to separate crudely, and an aliquot taken from the aqueous layer and centrifuged, at 5000 rpm for 5 min, to achieve complete separation. UV absorbance was determined using a Heios spectrophotometer at λ = 500–190 nm and a standard calibration curve prepared from serially diluted 10 mg of RA-AsA in 100 cm3 of mobile phase (see Section 2.10). The same procedure was repeated for each compound with the exception of AsA where the standard solution was prepared by dissolving 10 mg of AsA in 100 cm3 of deionised water (Fujita et al., 1964). 2.6. Skin samples The skin was defrosted and the subcutaneous fat carefully trimmed, before 2 cm2 disks were excised using a cork borer. The specimens were stored overnight at 2–4 ◦ C in sealed aluminium foil. Heat separated membranes were prepared by immersion in distilled water at 60 ◦ C for 1 min, after which stratum corneum and epidermis were carefully removed from the dermis using forceps (Christophers and Kligman, 1963).
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2.7. Skin permeation method Prior to use the skin specimens were immersed in de-ionised water for ∼3 h for the heat-separated membranes and ∼5 h for the full thickness (Ablett et al., 1996, Howes et al., 1996). The skin membranes were then mounted on Franz-type diffusion cells, with nominal diffusion area of 1.5 cm2 and a receptor volume of 3.5 ml. Receptor compartments were filled with de-gassed vehicle (methanol and pH 4.8 PBS), and the cells dosed with 100 l of 2.5 mM, of each of RA-AsA, Rol-Pal, and AsA-Pal and the donor caps occluded. The receptor phase was maintained in a water bath set at 37 ± 0.5 ◦ C under total light exclusion. At appropriate time intervals samples 200 l of receptor solution was withdrawn and replaced with fresh solution. Experiments were repeated with a dosage of 1.25 mM of each permeant to ensure consistency of the resulting permeability coefficients. Replication was n = 6 and two further cells dosed with vehicle alone served as a control.
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2.5 g of ammonium acetate. For ascorbates, a mobile phase of methanol:water, 80:20, pH 3.5, 1% acetic acid, was used. An injection volume of 20 l was used for the retinoids and 30 l for the ascorbates. Retention times for RA-AsA and Rol-Pal were 6.95–7.04 and 4.1–4.3 min, respectively. The limits of detection were 11.6 ng ml−1 , for the retinoids, and 56 ng ml−1 , for the ascorbates. Calibration curves were linear between the concentrations of the minimum detectable amounts and 5 mg ml−1 in the respective mobile phases. 2.10. Data processing Amounts permeated were plotted as a function of time and the linear portion of the curve determined as flux at steady-state (Jss ). The permeability coefficient (kp ) was calculated as kp = Jss /c, where c is the initial concentration in the vehicle applied to donor phase. Statistical comparisons were made using Microsoft Excel and the level of significance was taken as P = <0.05 (Skoog et al., 1996).
2.8. Skin penetration method—tape stripping 3. Results and discussion After 24 h the diffusion cells were dismantled and the membranes recovered before being subjected to tape stripping using the general method reported by Heard et al. (2003a). The area penetrated was washed with the respective mobile phase (see Section 2.10) then tape-stripped using D-Squame discs, which were collected in multiples of three and placed in 5 ml amber vials. The remaining tissue was cut into small pieces and placed in another vial and extracted with 2 ml of the respective mobile phase overnight using a mechanical rocker. Aliquots of 1 ml were centrifuged at 12,000 × g for 5 min, filtered and added to 1 ml amber HPLC auto-sampler vials. 2.9. Reverse phase HPLC analysis Samples were analysed at ambient temperature using an Agilent 1100 series automated HPLC system, fitted with a Kingsorb 5m 450 mm × 25 mm column (Phenomenex, Macclesfield, UK). The wavelengths used to detect the retinoids and ascorbates were 320 and 260 nm, respectively. The mobile phase for the retinoids consisted of acetonitrile:water:methanol (75:20:5) with buffer of pH 5.8, 1 ml formic acid and
3.1. Synthesis of retinyl ascorbate—RA-AsA The ester linkage was confirmed by placing 50 mg of the product in 2 ml of saturated ethanolic solution of hydroxylamine hydrochloride and 2 ml of 20% ethanolic potassium hydroxide. The mixture was heated to boiling point and acidified with 5% hydrochloric acid. Then, a 5% solution of ferrous chloride was added dropwise until the colour of deep red-purple developed, indicative of a positive result (Harwood et al., 1998). 1 HNMR (CD3 OD, 300 MHz), δ (in ppm) 1.04 (s. C16 and C17, 6H), 1.47 (m, C2, 2H), 1.6 (m, C3, 2H), 2.02 (C4, 2H) 1.71 (s, C18, 3H), 1.97 (s, C19, 3H), 2.33 (s, C20, 3H), 5.75 (s, C14, 1H), 6.25 (dd, C12, 1H), 6.95 (dd, C11, 1H), 6.15 (dd, C10, 1H), 6.13 (m, C8, 1H), 6.22 (m, C7, 1H), 4.16 (dd, C6 a, 1H), 4.23 (dd, C6 b, 1H), 4.11 (m, C5 , 1H), 4.79 (d, C4 , 1H); 13 CNMR; 33.5 (C1), 39.6 (C2), 18.8 (C3), 31.9 (C4), 130 (C5), 135.7 (C6), 137.2 (C9), 138.8 (C13), 137.4 (C7), 136 (C8), 130.3 (C10), 126.3 (C11), 125.6 (C12), 125.15 (C14), 27.9 (C16 and C17), 20.7 (C18), 12.5 (C19 and C20), 64.08 (C6 ), 65.14 (C5 ), 75.3
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(C4 ), 158.59 (C3 ), 117.33 (C2 ), 166.2, 173 (C=O). HPLC retention time, Rt = 6.95–7.035 min (mobile phase of acetonitrile:water:methanol, 75:20:5, buffer of pH 5.2–5.8, 1 ml formic acid and 2.5 g of ammonium acetate), purity > 98.5%; TLC, Rf = 3.21 (PEt:EtO:CHCl3, 1:2:1); ultra-violet spectra, wavelength λmax = 260–435 nm in mobile phase; molar decadic coefficient (ε) = 52,000 m−1 mol−1 at a standard of 0.658 mM; melting point (mp) = 183.5–185 ◦ C; calculated elemental analysis, C 68.1, H 7.47, O 24.2; experimental elemental analysis, C 68.03, H 7.32, O 24.4; MS (MeOH, HPLC grade) found 458.01 (M−• ): major signal m/z = 457.11, minor signal 1 m/z = 284.34, minor signal 2 m/z = 175.22. 3.2. Synthesis of ascorbyl palmitate (AsA-Pal) Ester linkage was confirmed as above. 1 HNMR (CD3 OD, 300 MHz), δ (in ppm) 0.98 (t, C16, 3H), 1.28 (m, C4-C15, 24H), 1.66 (m, C3, 2H), 2.32 (t, C2, 2H), 4.14 (dd, C6 a, 1H), 4.24 (dd, C6 b, 1H), 4.1 (m, C5 , 1H), 4.82 (d, C4 , 1H); 13 CNMR, 33.4, 31.26, 28.98 × 4, 28.95 × 2, 28.87, 28.68, 28.66, 28.45, 24.18, 22, 13.79 (C2-C16), 64.08 (C6 ), 65.14 (C5 ), 75.3 (C4 ), 158.59 (C3 ), 117.33 (C2 ), 166.8, 172.5 (C=O). HPLC retention time, Rt = 4.1–4.3 min (mobile phase, Me:water, 80:20, pH 3.5, 1% acetic acid), purity >99%; TLC, Rf = 3.1 (Pet:EtO:MeOH, 1:3:2); UV spectrum, wavelength λmax = 230–270 nm in mobile phase; molar decadic coefficient (ε) =17,000 m−1 mol−1 ; mp = 166–167 ◦ C; molecular weight (MW) = 414.53; calculated elemental analysis, C 63.74, H 9.34, O 27.02; experimental elemental analysis, C 63.56, H 9.16, O 27.3. An ester bond is the most convenient form of conjugation of chemical species for dermal delivery, because such a linkage would be expected to be susceptible to hydrolysis within the skin either enzymically, or chemically, or both (Sloan, 1992; Bonina et al., 2001). Usually, esterification reactions are acid catalysed by reacting acid and an alcohol, and the speed of the reaction depends on the capability of the catalysing acid and the reflux efficiency, when the chemical species are reacted together by the SN 2 mechanism. However, the esterification of a stronger acid (AsA) and a weaker acid (RA) in addition to the inherent instability of the two species at high temper-
atures required the removal of H2 O from the reaction and necessitated the use of a strong base resin for the removal of Cl− from the reaction mixture (Dowex, 2004). Ethanol was also used, since ethanol is neither protic nor aprotic at low temperatures (UWEC, edu., 2002) to force an SN 2 reaction. 3.3. Physicochemical determinants Table 1 summarises the physicochemical properties of the test compounds. The properties of the study compound, RA-AsA, are comparable with those of the commercially used Rol-Pal and AsA-Pal. The experimentally determined log Po/w values were approximate to the calculated log P (calculated using CS Chem Office). log P, is generally used to predict uptake into the stratum corneum (Kitagawa et al., 1997). Bando et al. (1997) reported the importance the dermal bioconversion rate as well as the lipophilicity of prodrugs development. A log P value of ∼ 2.2, for RA-AsA, indicates an affinity for the organic layer, which is mainly reflecting the RA as the lipophilic group, as demonstrated by the higher log P values for Rol and RA. It is considered that a 2 < log Po/w < 3 is optimal for topical delivery, however, such considerations are more suited for smaller chemical molecules with less polar side chains (Barlow, 1980; Schaefer and Redelmeier, 1996). The importance of a vehicle in percutaneous absorption is well documented (Sintov et al., 2003). RA-AsA exhibited low solubility and stability in water and had minimal stability, which was the same in aqueous ethanol and aqueous methanol. Some by-products that could be recognised in these solvents were the isomers 9-cis and 11-cis-retinoic acid—no AsA could be detected (data not shown). The aim was to have an approximate and consistent concentration of each test compound throughout the experiment time. Thus, Me:PBS, 50/50 pH 4.8 was selected as the vehicle, which was determined experimentally as the most favourable for the experiment. In the vehicle chosen, the zero-order decomposition rate of RA-AsA was 0.0032 mg ml−1 h−1 . This was comparable to those of Rol-Pal (0.002 mg ml−1 h−1 ) and AsA-Pal (0.0032 mg ml−1 h−1 ), over a period of 56 h. Table 1 also demonstrates that RA-AsA is substantially more stable than RA and AsA in the chosen vehicle and under our experimental conditions. The
± 2 ± 2
The values, for melting point (mp), solubility, and log Po/w are the mean ± S.D., n = 6. The decomposition constant is the slope obtained from plotting A0 − A against time (t), where A0 and A are the initial and that at time t concentrations, respectively. a Permeation coefficient, K = J /C , where J and C are the steady state flux, the slope of the straight-line part, and the applied concentration, respectively, n = 3 for each concentration. Permeability for AsA, RA p ss i ss i and Rol could not be determined because of their instability. b Calculations were made using the CS Chem-Draw Ultra.
Not determined Not determined Not determined 9.0 3.0 0.50 Not determined Not determined Not determined 262 ± 56 71 ± 35 12.5 ± 2 Not determined Not determined Not determined 9.0 3.0 2.10 Not determined Not determined Not determined 500.0 ± 75 139.1 ± 24 98.0 ± 12 −3.4 4.6 4.7 11 2.9 1.8 −1.9 ± 0.2 3.9 ± 0.3 4.3 ± 0.3 10.3 ± 0.2 1.9 ± 0.2 2.2 ± 0.3 0.08 0.1 0.05 0.002 0.003 0.0032 0.041 0.029 0.027 0.001 0.0024 0.0027 0.43 ± 0.05 0.45 ± 0.05 0.4 ± 0.01 0.56 ± 0.02 0.47 ± 0.02 0.46 ± 0.01 ± 1 ± 1 ± 1
124 146 110 Oil 172 187 AsA RA Rol Rol-PAL AsA-Pal RA-AsA Ascorbic acid All-trans-retinoic acid All-trans-retinol All-trans-retinyl palmitate Ascorbyl palmitate All-trans-retinyl ascorbate
176 300 286 525 415 458
Dose 1.25 mM
Jss ng cm−2 h−1 Kp × 10−3 (cm h−1)
Dose: 2.5 mM
Jss (ngcm−2 h−1 ) Experimental 37 ◦ C
Solubility Me:PBS (pH 4.8) (mg ml−1 ) (n ± S.D.) Meting point (◦ C) (n ± S.D.) MW Abbreviation Compound
Table 1 Physicochemical properties and summary of skin permeation data
Decomposition constant, k (mgml−1 h−1 ) in Me:PBS (pH 4.8) 22 ◦ C
log Po/w (n ± S.D.)
Calculatedb
Permeation through human membrane (n ± S.D.)a
Kp × 10−3 (cmh−1 )
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decomposition by-products from RA-AsA in this vehicle were determined as predominantly all-trans-RA and some AsA, by HPLC analysis. 3.4. Skin permeation It is known that retinoids generally penetrate the skin very poorly (Gollnick and Dummler, 1997) and consequently, a number of derivatives have been synthesised for use in the cosmetic industry, including Rol-Pal (Boehnlein et al., 1994) which possesses improved topical delivery properties (Lupo, 2001). Generally, topical formulations of RA and Rol sometimes contain a number of complex enhancers, UV-filters, and stabilisers. AsA-Pal has also been used for cosmetic applications (Kato et al., 1991). Unfortunately, RA, Rol, and AsA could not be detected in our skin experiments, due to their rapid decomposition under our experimental conditions. However, all-trans-RA-AsA, under the same experimental conditions, demonstrated favourable skin penetration characteristics, reaching steady state within 4 h with a flux (98 ng cm−2 h−1 ) at 2.5 mM concentration (Table 1; Figs. 2 and 3). However, this was lower than that for AsA-Pal, which was 139.1 and 500 ng cm−2 h−1 for Rol-Pal. This may have been due to the greater stability and lipophilicity of these two compounds. The permeability coefficients were 2.1, 3 and 9 cm h−1 for RA-AsA, AsA-Pal, and Rol-Pal, respectively, at 2.5 mM. However, while the permeability coefficient for Rol-Pal and AsA-Pal remained approximately the same at 1.25 mM concentration, for RA-AsA it was much lower, 0.5 cm h−1 , suggesting RA-AsA interacted with the skin in a concentration-dependent manner. Workers have demonstrated that the barrier function of the stratum corneum is unlikely to be compromised when treated with a vehicle of pH 3.5–8.5 (Sznitowska et al., 2001). It is also well-known that solvents can damage the barrier function of the stratum corneum, either by delipidising (Tsai et al., 2001) or lipid solubilization (Pershing et al., 1992). The effects of the Me:PBS vehicle on the barrier function of the stratum corneum was investigated in a separate study. The data indicated a degree of lipid solubilization, thus the penetration of the test compound would have been enhanced by the vehicle. It is recognised that the vehicle used does not represent a useable formulation and
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Rol-Pal
AsA-Pal
RA-AsA
9000
Permeation ng/cm2
8000 7000 6000 5000 4000 3000 2000 1000 0 0
5
10
15
20
Fig. 2. Comparison of permeation profiles for RA-AsA, Rol-Pal and AsA-Pal across human skin.
Fig. 3. Steady-state permeation plots for the determination of flux and permeability coefficient.
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Table 2 Summary of tape stripping results Phase
Amount of penetrant ng ( ± S.D.) RA-AsA
Donor SC VE Dermis Receptor
Rol-Pal
Ester
RA
AsA
Ester
29801 ± 1005 2914 ± 201 910 ± 190 92 ± 13 Not determined
Not determined Not determined 1004 ± 107 82 ± 15 Not determined
Not determined Not determined 644 ± 121 69 ± 10 Not determined
12656 1146 735 56 32067
that data may be influenced to some extent by these processes. 3.5. Tape stripping Table 2 and Fig. 4 illustrate the penetration of RA-AsA, Rol-Pal and AsA-Pal into full thickness human skin. Some 13% of RA-AsA could be detected in the different layers of the skin, 6% in the stratum corneum, 2% in the viable epidermis and 0.2% in the dermis in the ester form, delivering 2% of all-trans-RA and 1.4% of AsA to the viable epidermis, 0.2 and 0.15% of all-trans-RA and AsA,
± ± ± ± ±
AsA-Pal
623 168 88 21 941
Rol
Ester
Not determined Not determined 495 ± 80 67 ± 16 Not determined
20774 2392 1346 69 675
AsA ± ± ± ± ±
2.3 213 176 23 101
Not determined Not determined 611 ± 108 62 ± 19 Not determined
respectively, to the dermis. It is likely that the low amounts recovered in the donor phase and in the skin was due to free-radical degradation of RA-AsA and its parent compounds (unpublished data). Approximately 5% of Rol-Pal could be detected in the different regions of human skin, 2, 1, and 0.1% of the ester in the stratum corneum, viable epidermis and dermis, respectively. However, 57% of the ester was detected in the receptor phase, delivering 0.9 and 0.1% of Rol to the viable epidermis and dermis, respectively. Although most of the applied dose was recovered in the donor phase (23%) and in the skin (60%) and receptor phase, what is not accounted
Fig. 4. Penetration profiles of each ester. The numbers on the x-axis represents three tape stripping vs. the retained amounts in (ng cm−2 ; n = 6, ±S.D.). The total amount retained is calculated as the ester less its hydrolysed components.
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Fig. 5. Hydrolysis of penetrants in human skin as a function of depth.
for may have been lost due to degradation in the donor phase and the palmitate part of the hydrolysed ester. As for AsA-Pal, ∼10% could be detected in the different layers of the skin, 5, 3.0 and 0.15% of the ester form, in the stratum corneum, viable epidermis and dermis, respectively, delivering 1.3 and 0.1% of AsA to the viable epidermis and dermis, respectively, with 1.4% in the ester form was recovered from the receptor phase. The amounts of AsA-Pal that could be accounted for were, 44% in the donor phase, 10% in the skin and receptor, the large discrepancy is more than likely due to the degradation of AsA to an unidentified species. The characterisation of dermal esterase was reported by Boehnlein et al. (1994). The point at which the esters were hydrolysed in the skin was taken to be the uppermost layers of the viable epidermis: this was after 24 tape strips. The boundary between the viable epidermis and dermis was estimated at 42+ tape strips. Fig. 5 shows that the hydrolysis of RA-AsA appeared to commence at higher levels in the skin than Rol-Pal and AsA-pal, possibly as a result of differing penetration pathways. Alternatively, RA-AsA may be more susceptible to enzyme hydrolysis. The results demonstrated that RA-AsA was more extensively retained in the skin compared to Rol-Pal and AsA-Pal, which may be beneficial in the forma-
tion of a reservoir and limited systemic uptake. Both RA-AsA and AsA-Pal contain ascorbyl moieties with three free OH groups and would thus be expected to interact similarly with skin (Pugh et al., 1996; du Plessis et al., 2000) or vehicle (Heard et al., 2003b; Karia et al., 2004). An explanation for the difference in retentions may be that the retinoid hydrocarbon backbone in RA-AsA contains five conjugated double bonds with limited rotational flexibility, plus a ring structure at the end of the chain. This would affect their ability to form micelles, unlike AsA-Pal, which was shown to have the ability to form micelles in aqueous media (Nostro, 1997) and thus an enhanced rate of delivery (Bouwstra and Honeywell-Nguyen, 2002). Overall, the data in Table 2 and Fig. 5 indicated that RA-AsA is more labile to hydrolysis (chemical and/or enzymic) than the palmitate esters. Thus, RA-AsA can simultaneously deliver both parent moieties of the co-drug to the viable epidermis more effectively than the individual compounds from the palmitate prodrugs. Furthermore, use of RA-AsA would not give rise to palmitic acid residues which could have potentially toxic effects (Lima et al., 2002).
4. Conclusions An ester co-drug of all-trans-retinoic acid and l-ascorbic acid for dermal application has been
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prepared, which may have potential for treating UV-induced production of free radicals. It was found that the ester exhibited superior stability than either of the parent compounds and had virtually optimal log P for skin delivery. Compared to the commercially used all-trans-retinyl palmitate and ascorbyl palmitate esters, a greater degree of interaction with skin was observed which in practice, could beneficially limit the proportion of the dose entering the system. In addition, RA-AsA was found to be more labile to enzymatic/chemical hydrolysis than the palmitates, and following cleavage, potentially toxic residues would not be liberated as a byproduct.
References Ablett, S., Burdett, N.G., Adrian Carpenter, T., Hall, L.D., Salter, D.C., 1996. Short echo time MRI enables visualisation of the natural state of human stratum corneum water in vivo. Mag. Reson. Imag. 14, 357–360. Baggiolini, M., Boulay, F., Badwey, J.A., Cumute, J.T., 1993. Activation of neutrophil leukocytes: chemo-attractant receptors and respiratory burst. FASEB J. 7, 1004–1010. Bando, H., Sahashi, M., Yamashita, F., Takakura, Y., Hashida, M., 1997. Effects of skin metabolism on percutaneous penetration of lipophilic drugs. J. Pharm. Sci. 86, 759–761. Barlow, R.B., 1980. Quantitative Aspects of Chemical Pharmacology. Billing & Sons Ltd., pp.74–76. Boehnlein, J., Sakr, A., Lichtin, J.L., Bronaugh, R.L., 1994. Characterization of esterase and alcohol dehydrogenase in skin. Metabolism of retinyl palmitate to retinol (Vitamin A) during percutaneous absorption. Pharm. Res. 11, 1155–1159. Bonina, F.P., Puglia, C., Barbuzzi, T., de Caprariis, P., Palagiano, F., Rimoli, M.G., Saija, A., 2001. In vitro and in vivo evaluation of polyoxyethylene esters as dermal prodrugs of ketoprofen, naproxen and diclofenac. Eur. J. Pharm. Sci. 14, 123–134. Boreman, M.H., Napoli, J.L., 1996. Cellular retinol-binding protein-supported retinoid acid synthesis. J. Biol. Chem. 271, 301–309. Bouwstra, J.A., Honeywell-Nguyen, P.L., 2002. Skin structure and mode of action of vesicles. Adv. Drug Deliv. Rev. 54, S41–S55. Chikakane, K., Takahashi, H., 1995. Measurements of skin pH, and its significance in cutaneous diseases. Clin. Dermatol. 13, 299–306. Christophers, E., Kligman, A.M., 1963. Preparation of isolated sheets of human stratum corneum. Arch. Derm. 88, 702–704. Dalle Carbonare, M., Pathak, M.A., 1992. Skin photosensitizing agents and the role of reactive oxygen species in photoaging. J. Photochem. Photobiol. B. 14, 105–124. Darr, D., Dunston, S., Faust, H., 1996. Effectiveness of antioxidants (vitamin C and E) with and without sunscreen as topical photoprotectants. Acta. Dermatol. Venereol. 76, 264–268. Dowex, www.dowex.com/liquidsteps.
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keratinocyte ICAM-1 expression: UV-induced inhibition of cytokine-induced ICAM-1 mRNA expression is transient, differentially restored for IFN versus TNF, and followed by ICAM-1 induction via a TNF␣-like pathway. J. Invest. Dermatol. 98, 923–928. Lima, T.M., Kanufre, C.C., Pompiea, C., Verlengia, R., Curi, R., 2002. Ranking the toxicity of fatty acids on Jurkat and Raji cells by flow cytometric analysis. Tox. In Vitro 16, 741– 747. Lupo, M.P., 2001. Antioxidants and vitamins in cosmetics. Clin. Dermatol. 19, 467–473. Nachbar, F.H., Korting, C., 1995. The role of Vitamin E in normal and damaged skin. J. Mol. Med. 73, 7–17. Nazemi, M.H., Brain, K.R., Heard, C.M. 2000. Design of bifunctional moieties with improved skin delivery potential combining dexamethasone and NSAIDs. In: Brain, K.R., Walters, K. (Eds.), Perspectives in Percutaneous Penetration, vol. 7. STS Publishing, Cardiff, 67 pp. Nostro, P.L., 1997. Supramolecular aggregates from Vitamin C derivatives: structure and properties. Internet J. Sci. Biol. Chem.. Pence, B.C., Naylor, M.F., 1990. Effects of single-dose UV radiation on skin superoxide dismutase, catalase, and xanthine oxidase in hairless mice. J. Invest. Dermatol. 95, 213–216. Perchellet, J.P., Perchellet, E.M., 1989. Antioxidants and multistage carcinogenesis in mouse skin. Free Radic. Biol. Med. 7, 377– 408. Peus, D., Vasa, R.A., Meves, A., Pott, M., Beyerle, A., 1988. H2 O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes. J. Invest. Dermatol. 110, 966–971. Philips, C.L., Combs, S.B., Pinnell, S.R., 1994. Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human epidermal fibroblasts. J. Invest. Dermatol. 130, 228–232. Pershing, L.K., Silver, B.S., Kreuger, G.G., Shah, V.P., Skelly, J.P., 1992. Feasibility of measuring the bioavailability of topical betamethasone dipropionate in commercial formulations using drug content in skin blanching assay. Pharm. Res. 9, 45– 51. Pinnell, S.R., 1987. Induction of collagen synthesis by ascorbic acid. Arch. Dermatol. 123, 1684–1686.
Pugh, W.J., Roberts, M.S., Hadgraft, J., 1996. Epidermal permeability–penetrant structure relationships: 3. The effect of hydrogen bonding interactions and molecular size on diffusion across the stratum corneum. Int. J. Pharm. 138, 149–165. Schaefer, H., Redelmeier, T.E., 1996. Skin Barrier, Principles of Percutaneous Asorption. Karger, Switzerland, 214 pp. Shindo, Y., Witt, E., Packer, L., 1993. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light. J. Invest. Dermatol. 100, 260– 265. Shindo, Y., Witt, E., Han, D., Epstein, W., Packer, L., 1994. Enzymic and nonenzymic antioxidants in epidermis and dermis of human skin. J. Invest. Dermatol. 102, 122–124. Sintov, A.C., Krymberk, I., Gavrilov, V., Gorodischer, R., 2003. Transdermal delivery of paracetamol for paediatric use: effects of vehicle formulations on the percutaneous penetration. J.Pharm. Pharmacol. 55, 911–919. Skoog, D.A., West, D.M., Holler, F.J. 1996. Fundamentals of Analytical Chemistry, VII. Saunders College Publishing, USA, pp. 47–68. Sloan, K.B., 1992. Prodrugs: Topical and Ocular Drug Delivery. Marcel Decker, New York, pp. 46–32. Sznitowska, M., Janicki, S., Baczek, A., 2001. Studies on the effect of pH on the lipoidal route of penetration across stratum corneum. J. Control. Rel. 76, 327–335. Tsai, J., Sheu, H., Hung, P., Cheng, C., 2001. Effect of barrier disruption by acetone treatment on the permeability of compounds with various lipophilicities: implications for the permeability of compromised skin. J. Pharm. Sci. 90, 1242– 1254. UWEC, edu., 2002. www.uwec.edu/Academic/Curric/lewisd/ substitution/substitution.htm. Walter, K., Kurz, H., 1988. Binding of drugs to human skin: influencing factors and the role of tissue lipids. J. Pharm. Pharmacol. 40, 689–693. Weber, C., Podda, M., Rallis, M., Thiele, J.J., 1997. Efficacy of topical applied tocopherols and tocotrienols in protection of murine skin from oxidative damage induced by UV-irradiation. Free Rad. Biol. Med. 22, 761–769. Wilson, R.J., 2001. A thermodynamic exploration into pharmaceutical drug solubility. Drug Discov. Today 6, 985– 986.
Topical delivery of retinyl ascorbate co-drug 1. Synthesis, penetration into and permeation across human skin Kasem Abdulmajed, Charles M. Heard∗ Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK Received 21 October 2003; received in revised form 15 April 2004; accepted 10 May 2004
Abstract A novel synthetic technique was used to synthesise the co-drug retinyl ascorbate (RA-AsA) ester from all-trans-retinyl chloride (RA) and l-ascorbic acid (AsA) suspended in ethanol at low temperature. Its log P, solubility in a Me:PBS, 50/50 at pH 4.8 and degradation constant were determined. The flux and permeation coefficient were determined using heat separated human skin membrane, and skin penetration was determined by tape stripping using full thickness human. All experiments were performed in parallel with retinyl palmitate (Rol-Pal) and ascorbyl palmitate (AsA-Pal), which are used in commercial topical formulations. RA-AsA exhibited favourable log P (2.2), with stability much greater than RA and AsA, but similar stability to Rol-Pal and AsA-Pal. The flux of RA-AsA was lower than for Rol-Pal and AsA-Pal. RA-AsA also demonstrated higher skin retention than the other two esters, but delivered more RA and AsA to the viable epidermis than retinol from Rol-Pal and ascorbic acid from AsA-Pal. Overall, the data suggest the potential value of RA-AsA co-drug for the purpose of treating damage to skin resulting from UV-induced production of free radicals. © 2004 Elsevier B.V. All rights reserved. Keywords: Co-drug; Retinoic acid; Retinyl ascorbate; Anti-oxidant; Skin permeation; Tape stripping; Ascorbic acid
1. Introduction Epidermal keratinocytes are exposed to external and internal oxidative stress. Ultraviolet irradiation induces the generation of reactive oxygen species (ROS) and the lipid peroxidation of the plasma membrane (Krutmann et al., 1992). Physiological levels of ultraviolet B induce intracellular hydrogen peroxide (H2 O2 ) generation in human epidermal keratinocytes (Peus et al., 1988). Infiltrating polymorphonuclear leukocytes and macrophages also generate ROS including superoxide anion radical, H2 O2 and hydroxyl ∗ Corresponding author. Tel.: +44 29 20875819; fax: +44 29 20875819. E-mail address: [email protected] (C.M. Heard).
radical (Baggiolini et al., 1993; Ishii et al., 1999). ROS are implicated in ultraviolet UV-light induced damage to skin (Perchellet and Perchellet, 1989) which could also be an initiator in the pathogenesis of photoaging and skin cancer (Dalle Carbonare and Pathak, 1992; Emerit, 1992; Shindo et al., 1994; Nachbar and Korting, 1995). Antioxidants that protect the skin against ROS include various low molecular weight antioxidants (glutathione, tocopherol and ubiquinol) and antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and thioredoxin reductase) (Fuchs et al., 1989, Pence and Naylor, 1990; Shindo et al., 1993; Nachbar and Korting, 1995). Topical administration of antioxidants provides an efficient way to enrich the endogenous cutaneous protection system and thus may be a successful strategy
0378-5173/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2004.05.008
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for diminishing ultraviolet radiation-mediated oxidative damage in the skin (Khettab et al., 1988; Weber et al., 1997). Retinoic acid (RA) is highly lipophilic (log P = 4.6) and the most potent form of the retinoids, which exert a wide variety of effects in the regulation of cell division, and possess some antioxidant properties (Khettab et al., 1988; Boreman and Napoli, 1996). l-Ascorbic acid (AsA) is a strong and powerful water-soluble antioxidant that efficiently protects important organic and biological molecules against ox-
idative degradation by inhibiting free radical initiated lipid peroxidation, a process believed to be responsible for photoaging and the induction of collagen synthesis (Pinnell, 1987; Philips et al., 1994). It functions even better in conjunction with other antioxidants, such as Vitamin E (tocopherols) and carotenoids, by establishing a peculiar recycling system with a synergic effect (Darr et al., 1996). However, due to the very low solubility of AsA in non-aqueous media (log P = −3.4), it is not well-suited to dermal application. In addition,
Fig. 1. Chemical structures of the test compounds.
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both RA and AsA are highly unstable when exposed to light, air and elevated temperatures. RA contains a carboxylic acid group, whereas AsA contains hydroxyl functionality, thus allowing the formation of an ester co-drug. Co-drugs were first described by Jones (1990), although the first report of a co-drug for topical application was by Nazemi et al. (2000). It is hypothesised that the co-drug retinyl ascorbate (RA-AsA) would demonstrate enhanced dermal uptake properties relative to both parent moieties as it would be more lipophilic (log P = 2.2) and be subject to greater uptake into the lipoidal domains of the stratum corneum (Walter and Kurz, 1988). Furthermore, the ester linkage could confer greater stability to both parent compounds, allowing them to penetrate deeper into the skin and provide combined regulatory/antioxidant properties, once the ester bond is hydrolysed in situ, either enzymatically or chemically. Thus, dermal application should be enhanced for protection against UV-induced oxidative damage to the skin. In this study RA-AsA co-drug was synthesised and its solubility and stability in different vehicles determined, before being studied in vitro for its suitability for application to human skin. For comparative purposes, retinyl palmitate (Rol-Pal) and ascorbyl palmitate (AsA-Pal), both of which are used in topical formulations for cosmetic and medicinal purposes (Boehnlein et al., 1994; Darr et al., 1996) were also examined. The structures of the compounds studied in the work are shown in Fig. 1.
2. Materials and methods 2.1. Materials All-trans-retinoic acid (RA), l-ascorbic acid (AsA), all-trans-retinol (Rol), all-trans-retinol palmitate (Rol-Pal), palmitic acid (all of purity > 99), phosphorous trichloride and OH-substituted anion exchange resin were obtained from Sigma (Poole, UK). Ultrafree-CL 2 ml tubes, containing low-binding Durapore PVDF 0.22 m filter inserts, were from Millipore (Bedford, MA, USA). D-Squame skin sampling discs, 14 mm, were from CuDerm Corporation (Dallas, Texas, USA). All other reagents and solvents were from Fisher (Loughborough, UK). Female
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abdominal human skin was obtained post-cosmetic surgery and frozen immediately. 2.2. Synthesis of retinyl ascorbate Firstly, retinyl chloride was synthesised. A solution of 1 mM phosphorous trichloride in 6 ml of dry benzene was added slowly to a stirring suspension of 1.5 g (1.4 mM) of all-trans-retinoic acid in15 ml of benzene and stirred continuously at 25 ◦ C, for 1 h. The reaction mixture was then allowed to cool in an ice bath and was the mixture decanted to separate the precipitate. The solution was then tested for the presence of chloride by reacting 0.5 ml with an ethanolic solution of silver nitrite and potassium iodide yielding a white precipitate of silver chloride. The reaction was followed by TLC, Rf = 0.2 in (Pet:EtO:CHCl3 ), (1:1:1) (Fulmer Shealy et al., 1988). l-Ascorbic acid (1.7 g) was suspended in 30 ml of ethanol and allowed to stand over 2 g of molecular sieves and allowed to dry overnight under light-shield. The sieves were removed and 2 g of dry anion exchange resin was added to the solution. After 30 min, 10 mg of K2 CO3 were added the reaction and 15 ml of retinyl chloride added, drop wise, and the mixture stirred overnight at 30 ◦ C and under light-shield. The reaction was followed by TLC (silica gel/1:2:1 Pet:EtO:CHCl3 ). After 15 h the solvent was evaporated under vacuum and the concentrate dissolved in 250 ml of CH3 OH:CHCl3 and washed with 5 M brine. Two hundred and fifty milliliters of n-hexane was added and cooled to 4 ◦ C. The lower layer was concentrated and purified three times by column chromatography to obtain 264 mg of product in the form of an off-white powder and with a purity of >98.5%. 2.3. Synthesis of ascorbyl palmitate Firstly, palmityl chloride was synthesised. A solution of 2.3 g (0.84 M) of phosphorous trichloride in 20 ml of dry benzene was added slowly to a stirring suspension of 15 g (1.4 M) of palmitic acid in 50 ml of benzene. Stirring was continued, at 25 ◦ C, for 1 h. The reaction mixture was then allowed to cool and then decanted to separate the precipitate. The solution was then tested for the presence of chloride by reacting 0.5 ml with an ethanolic solution of silver nitrite
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and potassium iodide yielding a white precipitate of silver chloride. Ascorbic acid (10 mg) was added to a solution of 50 ml of palmityl chloride in 150 ml of pyridine. The mixture was then stirred for 40 h at 40 ◦ C, under light-shield, and nitrogen. The reaction was followed by TLC (silica gel; PEt:EtO:MeOH, 1:3:2). The reaction mixture was then concentrated under pressure. Ethyl acetate was added to the oil residue, which immediately gave a white precipitate. After filtering, the residue was washed with hot EtOAc and hot water (60–70 ◦ C) and then dried. This was then recrystallized from EtOH:H2 O (4:1) to give 467 mg of white powder. 2.4. Solubility and stability issues AsA was highly soluble in water but of limited solubility in ethanol, whereas RA-AsA, RA, Rol, Rol-Pal, and AsA-Pal are insoluble in water but of varying solubility amounts in ethanol. However, all the compounds are soluble in methanol, but with limited stability. Furthermore, the RA-AsA, RA and AsA exhibited limited stability in an ethanol/water, and methanol/water co-solvents. It was determined (data not shown) that a co-solvent system of Me:PBS, 50:50, pH 4.8, was the most suitable for all six compounds in terms of solubility and stability for the experiment duration. Also, pH 4.8 is near to the pH of human skin surface of ∼5.5. AsA, RA, Rol and RA-AsA are highly unstable on exposure to light, temperature and atmospheric oxygen (Chikakane and Takahashi, 1995). Therefore, their solubility was determined by the shaking and assaying method, i.e. on the basis of the compounds kinetics rather than their thermodynamics. Employing the kinetic method (shaking then assaying) for the thermodynamic method (shaking until equilibrium) traded the uncertainty of a desired form for the certainty of a potentially undesired form (Wilson, 2001). 2.4.1. Solubility An excess of each compound was placed in Ultrafree-CL tubes containing 1 ml solution of methanol and PBS, pH 4.8. The vials were then placed on a blood tube rotator in an incubation oven set at 37 ◦ C for 1 h under light-shield, the tubes were then centrifuged at 12,000 rpm for 5 min, 100 l samples were, then assayed by HPLC (section 2.10.), the ex-
periments were carried out in triplicates, the remainder was used to determine the calibration curve and the lowest detection limit and for the skin experiments. 2.4.2. Stability A 5 ml saturated solution of each compound in Me/PBS was added to glass vials in six sets of x3. Two sets were maintained at room temperature, 37 and 60 ◦ C whilst stirring. One set was shielded from light, the other not. Samples (100 l) were collected from each vial at regular intervals over a period of 56 h. Experiments were terminated at 1 h (for the 60 ◦ C for both shielded and unshielded), 1 h (for the 37 ◦ C unshielded), and 2 h (for those placed at room temperature, unshielded). The samples were then analysed by HPLC. Zero order decomposition rates, k (mg ml−1 h−1 ), were calculated from the equation: A0 − A = kt, where A0 is the initial concentration, A is the remaining concentration at time t, and k is the gradient of [A0 − A] against t. 2.5. log Po/w determination Samples (∼60 mg) were placed in octanol:deionised water (50:150 cm3 ) in duplicates and shaken vigourously on a mechanical shaker, for 30 min. The layers were allowed to separate crudely, and an aliquot taken from the aqueous layer and centrifuged, at 5000 rpm for 5 min, to achieve complete separation. UV absorbance was determined using a Heios spectrophotometer at λ = 500–190 nm and a standard calibration curve prepared from serially diluted 10 mg of RA-AsA in 100 cm3 of mobile phase (see Section 2.10). The same procedure was repeated for each compound with the exception of AsA where the standard solution was prepared by dissolving 10 mg of AsA in 100 cm3 of deionised water (Fujita et al., 1964). 2.6. Skin samples The skin was defrosted and the subcutaneous fat carefully trimmed, before 2 cm2 disks were excised using a cork borer. The specimens were stored overnight at 2–4 ◦ C in sealed aluminium foil. Heat separated membranes were prepared by immersion in distilled water at 60 ◦ C for 1 min, after which stratum corneum and epidermis were carefully removed from the dermis using forceps (Christophers and Kligman, 1963).
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2.7. Skin permeation method Prior to use the skin specimens were immersed in de-ionised water for ∼3 h for the heat-separated membranes and ∼5 h for the full thickness (Ablett et al., 1996, Howes et al., 1996). The skin membranes were then mounted on Franz-type diffusion cells, with nominal diffusion area of 1.5 cm2 and a receptor volume of 3.5 ml. Receptor compartments were filled with de-gassed vehicle (methanol and pH 4.8 PBS), and the cells dosed with 100 l of 2.5 mM, of each of RA-AsA, Rol-Pal, and AsA-Pal and the donor caps occluded. The receptor phase was maintained in a water bath set at 37 ± 0.5 ◦ C under total light exclusion. At appropriate time intervals samples 200 l of receptor solution was withdrawn and replaced with fresh solution. Experiments were repeated with a dosage of 1.25 mM of each permeant to ensure consistency of the resulting permeability coefficients. Replication was n = 6 and two further cells dosed with vehicle alone served as a control.
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2.5 g of ammonium acetate. For ascorbates, a mobile phase of methanol:water, 80:20, pH 3.5, 1% acetic acid, was used. An injection volume of 20 l was used for the retinoids and 30 l for the ascorbates. Retention times for RA-AsA and Rol-Pal were 6.95–7.04 and 4.1–4.3 min, respectively. The limits of detection were 11.6 ng ml−1 , for the retinoids, and 56 ng ml−1 , for the ascorbates. Calibration curves were linear between the concentrations of the minimum detectable amounts and 5 mg ml−1 in the respective mobile phases. 2.10. Data processing Amounts permeated were plotted as a function of time and the linear portion of the curve determined as flux at steady-state (Jss ). The permeability coefficient (kp ) was calculated as kp = Jss /c, where c is the initial concentration in the vehicle applied to donor phase. Statistical comparisons were made using Microsoft Excel and the level of significance was taken as P = <0.05 (Skoog et al., 1996).
2.8. Skin penetration method—tape stripping 3. Results and discussion After 24 h the diffusion cells were dismantled and the membranes recovered before being subjected to tape stripping using the general method reported by Heard et al. (2003a). The area penetrated was washed with the respective mobile phase (see Section 2.10) then tape-stripped using D-Squame discs, which were collected in multiples of three and placed in 5 ml amber vials. The remaining tissue was cut into small pieces and placed in another vial and extracted with 2 ml of the respective mobile phase overnight using a mechanical rocker. Aliquots of 1 ml were centrifuged at 12,000 × g for 5 min, filtered and added to 1 ml amber HPLC auto-sampler vials. 2.9. Reverse phase HPLC analysis Samples were analysed at ambient temperature using an Agilent 1100 series automated HPLC system, fitted with a Kingsorb 5m 450 mm × 25 mm column (Phenomenex, Macclesfield, UK). The wavelengths used to detect the retinoids and ascorbates were 320 and 260 nm, respectively. The mobile phase for the retinoids consisted of acetonitrile:water:methanol (75:20:5) with buffer of pH 5.8, 1 ml formic acid and
3.1. Synthesis of retinyl ascorbate—RA-AsA The ester linkage was confirmed by placing 50 mg of the product in 2 ml of saturated ethanolic solution of hydroxylamine hydrochloride and 2 ml of 20% ethanolic potassium hydroxide. The mixture was heated to boiling point and acidified with 5% hydrochloric acid. Then, a 5% solution of ferrous chloride was added dropwise until the colour of deep red-purple developed, indicative of a positive result (Harwood et al., 1998). 1 HNMR (CD3 OD, 300 MHz), δ (in ppm) 1.04 (s. C16 and C17, 6H), 1.47 (m, C2, 2H), 1.6 (m, C3, 2H), 2.02 (C4, 2H) 1.71 (s, C18, 3H), 1.97 (s, C19, 3H), 2.33 (s, C20, 3H), 5.75 (s, C14, 1H), 6.25 (dd, C12, 1H), 6.95 (dd, C11, 1H), 6.15 (dd, C10, 1H), 6.13 (m, C8, 1H), 6.22 (m, C7, 1H), 4.16 (dd, C6 a, 1H), 4.23 (dd, C6 b, 1H), 4.11 (m, C5 , 1H), 4.79 (d, C4 , 1H); 13 CNMR; 33.5 (C1), 39.6 (C2), 18.8 (C3), 31.9 (C4), 130 (C5), 135.7 (C6), 137.2 (C9), 138.8 (C13), 137.4 (C7), 136 (C8), 130.3 (C10), 126.3 (C11), 125.6 (C12), 125.15 (C14), 27.9 (C16 and C17), 20.7 (C18), 12.5 (C19 and C20), 64.08 (C6 ), 65.14 (C5 ), 75.3
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(C4 ), 158.59 (C3 ), 117.33 (C2 ), 166.2, 173 (C=O). HPLC retention time, Rt = 6.95–7.035 min (mobile phase of acetonitrile:water:methanol, 75:20:5, buffer of pH 5.2–5.8, 1 ml formic acid and 2.5 g of ammonium acetate), purity > 98.5%; TLC, Rf = 3.21 (PEt:EtO:CHCl3, 1:2:1); ultra-violet spectra, wavelength λmax = 260–435 nm in mobile phase; molar decadic coefficient (ε) = 52,000 m−1 mol−1 at a standard of 0.658 mM; melting point (mp) = 183.5–185 ◦ C; calculated elemental analysis, C 68.1, H 7.47, O 24.2; experimental elemental analysis, C 68.03, H 7.32, O 24.4; MS (MeOH, HPLC grade) found 458.01 (M−• ): major signal m/z = 457.11, minor signal 1 m/z = 284.34, minor signal 2 m/z = 175.22. 3.2. Synthesis of ascorbyl palmitate (AsA-Pal) Ester linkage was confirmed as above. 1 HNMR (CD3 OD, 300 MHz), δ (in ppm) 0.98 (t, C16, 3H), 1.28 (m, C4-C15, 24H), 1.66 (m, C3, 2H), 2.32 (t, C2, 2H), 4.14 (dd, C6 a, 1H), 4.24 (dd, C6 b, 1H), 4.1 (m, C5 , 1H), 4.82 (d, C4 , 1H); 13 CNMR, 33.4, 31.26, 28.98 × 4, 28.95 × 2, 28.87, 28.68, 28.66, 28.45, 24.18, 22, 13.79 (C2-C16), 64.08 (C6 ), 65.14 (C5 ), 75.3 (C4 ), 158.59 (C3 ), 117.33 (C2 ), 166.8, 172.5 (C=O). HPLC retention time, Rt = 4.1–4.3 min (mobile phase, Me:water, 80:20, pH 3.5, 1% acetic acid), purity >99%; TLC, Rf = 3.1 (Pet:EtO:MeOH, 1:3:2); UV spectrum, wavelength λmax = 230–270 nm in mobile phase; molar decadic coefficient (ε) =17,000 m−1 mol−1 ; mp = 166–167 ◦ C; molecular weight (MW) = 414.53; calculated elemental analysis, C 63.74, H 9.34, O 27.02; experimental elemental analysis, C 63.56, H 9.16, O 27.3. An ester bond is the most convenient form of conjugation of chemical species for dermal delivery, because such a linkage would be expected to be susceptible to hydrolysis within the skin either enzymically, or chemically, or both (Sloan, 1992; Bonina et al., 2001). Usually, esterification reactions are acid catalysed by reacting acid and an alcohol, and the speed of the reaction depends on the capability of the catalysing acid and the reflux efficiency, when the chemical species are reacted together by the SN 2 mechanism. However, the esterification of a stronger acid (AsA) and a weaker acid (RA) in addition to the inherent instability of the two species at high temper-
atures required the removal of H2 O from the reaction and necessitated the use of a strong base resin for the removal of Cl− from the reaction mixture (Dowex, 2004). Ethanol was also used, since ethanol is neither protic nor aprotic at low temperatures (UWEC, edu., 2002) to force an SN 2 reaction. 3.3. Physicochemical determinants Table 1 summarises the physicochemical properties of the test compounds. The properties of the study compound, RA-AsA, are comparable with those of the commercially used Rol-Pal and AsA-Pal. The experimentally determined log Po/w values were approximate to the calculated log P (calculated using CS Chem Office). log P, is generally used to predict uptake into the stratum corneum (Kitagawa et al., 1997). Bando et al. (1997) reported the importance the dermal bioconversion rate as well as the lipophilicity of prodrugs development. A log P value of ∼ 2.2, for RA-AsA, indicates an affinity for the organic layer, which is mainly reflecting the RA as the lipophilic group, as demonstrated by the higher log P values for Rol and RA. It is considered that a 2 < log Po/w < 3 is optimal for topical delivery, however, such considerations are more suited for smaller chemical molecules with less polar side chains (Barlow, 1980; Schaefer and Redelmeier, 1996). The importance of a vehicle in percutaneous absorption is well documented (Sintov et al., 2003). RA-AsA exhibited low solubility and stability in water and had minimal stability, which was the same in aqueous ethanol and aqueous methanol. Some by-products that could be recognised in these solvents were the isomers 9-cis and 11-cis-retinoic acid—no AsA could be detected (data not shown). The aim was to have an approximate and consistent concentration of each test compound throughout the experiment time. Thus, Me:PBS, 50/50 pH 4.8 was selected as the vehicle, which was determined experimentally as the most favourable for the experiment. In the vehicle chosen, the zero-order decomposition rate of RA-AsA was 0.0032 mg ml−1 h−1 . This was comparable to those of Rol-Pal (0.002 mg ml−1 h−1 ) and AsA-Pal (0.0032 mg ml−1 h−1 ), over a period of 56 h. Table 1 also demonstrates that RA-AsA is substantially more stable than RA and AsA in the chosen vehicle and under our experimental conditions. The
± 2 ± 2
The values, for melting point (mp), solubility, and log Po/w are the mean ± S.D., n = 6. The decomposition constant is the slope obtained from plotting A0 − A against time (t), where A0 and A are the initial and that at time t concentrations, respectively. a Permeation coefficient, K = J /C , where J and C are the steady state flux, the slope of the straight-line part, and the applied concentration, respectively, n = 3 for each concentration. Permeability for AsA, RA p ss i ss i and Rol could not be determined because of their instability. b Calculations were made using the CS Chem-Draw Ultra.
Not determined Not determined Not determined 9.0 3.0 0.50 Not determined Not determined Not determined 262 ± 56 71 ± 35 12.5 ± 2 Not determined Not determined Not determined 9.0 3.0 2.10 Not determined Not determined Not determined 500.0 ± 75 139.1 ± 24 98.0 ± 12 −3.4 4.6 4.7 11 2.9 1.8 −1.9 ± 0.2 3.9 ± 0.3 4.3 ± 0.3 10.3 ± 0.2 1.9 ± 0.2 2.2 ± 0.3 0.08 0.1 0.05 0.002 0.003 0.0032 0.041 0.029 0.027 0.001 0.0024 0.0027 0.43 ± 0.05 0.45 ± 0.05 0.4 ± 0.01 0.56 ± 0.02 0.47 ± 0.02 0.46 ± 0.01 ± 1 ± 1 ± 1
124 146 110 Oil 172 187 AsA RA Rol Rol-PAL AsA-Pal RA-AsA Ascorbic acid All-trans-retinoic acid All-trans-retinol All-trans-retinyl palmitate Ascorbyl palmitate All-trans-retinyl ascorbate
176 300 286 525 415 458
Dose 1.25 mM
Jss ng cm−2 h−1 Kp × 10−3 (cm h−1)
Dose: 2.5 mM
Jss (ngcm−2 h−1 ) Experimental 37 ◦ C
Solubility Me:PBS (pH 4.8) (mg ml−1 ) (n ± S.D.) Meting point (◦ C) (n ± S.D.) MW Abbreviation Compound
Table 1 Physicochemical properties and summary of skin permeation data
Decomposition constant, k (mgml−1 h−1 ) in Me:PBS (pH 4.8) 22 ◦ C
log Po/w (n ± S.D.)
Calculatedb
Permeation through human membrane (n ± S.D.)a
Kp × 10−3 (cmh−1 )
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decomposition by-products from RA-AsA in this vehicle were determined as predominantly all-trans-RA and some AsA, by HPLC analysis. 3.4. Skin permeation It is known that retinoids generally penetrate the skin very poorly (Gollnick and Dummler, 1997) and consequently, a number of derivatives have been synthesised for use in the cosmetic industry, including Rol-Pal (Boehnlein et al., 1994) which possesses improved topical delivery properties (Lupo, 2001). Generally, topical formulations of RA and Rol sometimes contain a number of complex enhancers, UV-filters, and stabilisers. AsA-Pal has also been used for cosmetic applications (Kato et al., 1991). Unfortunately, RA, Rol, and AsA could not be detected in our skin experiments, due to their rapid decomposition under our experimental conditions. However, all-trans-RA-AsA, under the same experimental conditions, demonstrated favourable skin penetration characteristics, reaching steady state within 4 h with a flux (98 ng cm−2 h−1 ) at 2.5 mM concentration (Table 1; Figs. 2 and 3). However, this was lower than that for AsA-Pal, which was 139.1 and 500 ng cm−2 h−1 for Rol-Pal. This may have been due to the greater stability and lipophilicity of these two compounds. The permeability coefficients were 2.1, 3 and 9 cm h−1 for RA-AsA, AsA-Pal, and Rol-Pal, respectively, at 2.5 mM. However, while the permeability coefficient for Rol-Pal and AsA-Pal remained approximately the same at 1.25 mM concentration, for RA-AsA it was much lower, 0.5 cm h−1 , suggesting RA-AsA interacted with the skin in a concentration-dependent manner. Workers have demonstrated that the barrier function of the stratum corneum is unlikely to be compromised when treated with a vehicle of pH 3.5–8.5 (Sznitowska et al., 2001). It is also well-known that solvents can damage the barrier function of the stratum corneum, either by delipidising (Tsai et al., 2001) or lipid solubilization (Pershing et al., 1992). The effects of the Me:PBS vehicle on the barrier function of the stratum corneum was investigated in a separate study. The data indicated a degree of lipid solubilization, thus the penetration of the test compound would have been enhanced by the vehicle. It is recognised that the vehicle used does not represent a useable formulation and
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Rol-Pal
AsA-Pal
RA-AsA
9000
Permeation ng/cm2
8000 7000 6000 5000 4000 3000 2000 1000 0 0
5
10
15
20
Fig. 2. Comparison of permeation profiles for RA-AsA, Rol-Pal and AsA-Pal across human skin.
Fig. 3. Steady-state permeation plots for the determination of flux and permeability coefficient.
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Table 2 Summary of tape stripping results Phase
Amount of penetrant ng ( ± S.D.) RA-AsA
Donor SC VE Dermis Receptor
Rol-Pal
Ester
RA
AsA
Ester
29801 ± 1005 2914 ± 201 910 ± 190 92 ± 13 Not determined
Not determined Not determined 1004 ± 107 82 ± 15 Not determined
Not determined Not determined 644 ± 121 69 ± 10 Not determined
12656 1146 735 56 32067
that data may be influenced to some extent by these processes. 3.5. Tape stripping Table 2 and Fig. 4 illustrate the penetration of RA-AsA, Rol-Pal and AsA-Pal into full thickness human skin. Some 13% of RA-AsA could be detected in the different layers of the skin, 6% in the stratum corneum, 2% in the viable epidermis and 0.2% in the dermis in the ester form, delivering 2% of all-trans-RA and 1.4% of AsA to the viable epidermis, 0.2 and 0.15% of all-trans-RA and AsA,
± ± ± ± ±
AsA-Pal
623 168 88 21 941
Rol
Ester
Not determined Not determined 495 ± 80 67 ± 16 Not determined
20774 2392 1346 69 675
AsA ± ± ± ± ±
2.3 213 176 23 101
Not determined Not determined 611 ± 108 62 ± 19 Not determined
respectively, to the dermis. It is likely that the low amounts recovered in the donor phase and in the skin was due to free-radical degradation of RA-AsA and its parent compounds (unpublished data). Approximately 5% of Rol-Pal could be detected in the different regions of human skin, 2, 1, and 0.1% of the ester in the stratum corneum, viable epidermis and dermis, respectively. However, 57% of the ester was detected in the receptor phase, delivering 0.9 and 0.1% of Rol to the viable epidermis and dermis, respectively. Although most of the applied dose was recovered in the donor phase (23%) and in the skin (60%) and receptor phase, what is not accounted
Fig. 4. Penetration profiles of each ester. The numbers on the x-axis represents three tape stripping vs. the retained amounts in (ng cm−2 ; n = 6, ±S.D.). The total amount retained is calculated as the ester less its hydrolysed components.
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Fig. 5. Hydrolysis of penetrants in human skin as a function of depth.
for may have been lost due to degradation in the donor phase and the palmitate part of the hydrolysed ester. As for AsA-Pal, ∼10% could be detected in the different layers of the skin, 5, 3.0 and 0.15% of the ester form, in the stratum corneum, viable epidermis and dermis, respectively, delivering 1.3 and 0.1% of AsA to the viable epidermis and dermis, respectively, with 1.4% in the ester form was recovered from the receptor phase. The amounts of AsA-Pal that could be accounted for were, 44% in the donor phase, 10% in the skin and receptor, the large discrepancy is more than likely due to the degradation of AsA to an unidentified species. The characterisation of dermal esterase was reported by Boehnlein et al. (1994). The point at which the esters were hydrolysed in the skin was taken to be the uppermost layers of the viable epidermis: this was after 24 tape strips. The boundary between the viable epidermis and dermis was estimated at 42+ tape strips. Fig. 5 shows that the hydrolysis of RA-AsA appeared to commence at higher levels in the skin than Rol-Pal and AsA-pal, possibly as a result of differing penetration pathways. Alternatively, RA-AsA may be more susceptible to enzyme hydrolysis. The results demonstrated that RA-AsA was more extensively retained in the skin compared to Rol-Pal and AsA-Pal, which may be beneficial in the forma-
tion of a reservoir and limited systemic uptake. Both RA-AsA and AsA-Pal contain ascorbyl moieties with three free OH groups and would thus be expected to interact similarly with skin (Pugh et al., 1996; du Plessis et al., 2000) or vehicle (Heard et al., 2003b; Karia et al., 2004). An explanation for the difference in retentions may be that the retinoid hydrocarbon backbone in RA-AsA contains five conjugated double bonds with limited rotational flexibility, plus a ring structure at the end of the chain. This would affect their ability to form micelles, unlike AsA-Pal, which was shown to have the ability to form micelles in aqueous media (Nostro, 1997) and thus an enhanced rate of delivery (Bouwstra and Honeywell-Nguyen, 2002). Overall, the data in Table 2 and Fig. 5 indicated that RA-AsA is more labile to hydrolysis (chemical and/or enzymic) than the palmitate esters. Thus, RA-AsA can simultaneously deliver both parent moieties of the co-drug to the viable epidermis more effectively than the individual compounds from the palmitate prodrugs. Furthermore, use of RA-AsA would not give rise to palmitic acid residues which could have potentially toxic effects (Lima et al., 2002).
4. Conclusions An ester co-drug of all-trans-retinoic acid and l-ascorbic acid for dermal application has been
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prepared, which may have potential for treating UV-induced production of free radicals. It was found that the ester exhibited superior stability than either of the parent compounds and had virtually optimal log P for skin delivery. Compared to the commercially used all-trans-retinyl palmitate and ascorbyl palmitate esters, a greater degree of interaction with skin was observed which in practice, could beneficially limit the proportion of the dose entering the system. In addition, RA-AsA was found to be more labile to enzymatic/chemical hydrolysis than the palmitates, and following cleavage, potentially toxic residues would not be liberated as a byproduct.
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