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Journal of Pharmacy
Research Paper
And Pharmacology
Keywords Abstract
a-tocopherol; a-tocopherol acetate;
polyunsaturated fatty acids; skin permeation Objectives The aim of this work was to investigate new pro-vitamins based on
a-tocopherol (a-Toc) and fatty acids, and to compare their properties with those
Correspondence of a-tocopherol acetate (a-TAc).
Amnon Sintov and Shimon Ben-Shabat, E.D.
Methods Skin levels of a-Toc–fatty acid ester conjugates, total a-Toc and endog-
Bergmann Campus, Ben-Gurion University of
the Negev, P.O. Box 653, Beer Sheva 84105,
enous a-Toc were measured in skin samples taken from separate groups of treated
Israel. and untreated rats. Multiple and extensive treatment with a-Toc oleate and a-TAc
E-mail: asintov@bgu.ac.il; sbs@bgu.ac.il was also carried out to assess the skin accumulation and safety of these esters.
Key findings The in-vivo studies revealed that a-Toc–fatty acid conjugates pen-
Received August 9, 2012 etrated into the skin quantitatively while being comparable with the permeation
Accepted December 10, 2012 of a-TAc. Differences were found between the levels of total a-Toc and endog-
enous a-Toc after application of a-TAc, a-Toc oleate, a-Toc linoleate, a-Toc-a
doi: 10.1111/jphp.12027
linolenate and a-Toc palmitate, indicating that a-Toc conjugates of these fatty
acids, but not a-Toc g-linolenate or a-Toc stearate, were hydrolysed to free
a-Toc. In long-term and extensive treatment, a-TAc was found to be lethal to rats
treated with 1.15 mg/kg of this agent, which had been spread over 16 cm2 of skin.
Similar treatment with a-Toc oleate did not produce any side effects.
Conclusions This study suggests that a-Toc conjugates with unsaturated fatty
acids may be a good alternative as stable vitamin E derivatives, rather than the
a-TAc ester.
Introduction
It has long been recognized that a-tocopherol (a-Toc) is the applied a-TAc have been examined in several studies;
major lipophilic antioxidant found in living tissues.[1] It is a however, it was found to be less effective than a-Toc and
radical-scavenging antioxidant that suppresses chain propa- showed no reduction in UV-induced radical flux in the
gation by donating its phenolic hydrogen to oxygen radi- skin, as compared with a-Toc.[9,10] Furthermore, a-TAc
cals.[2] It was shown in several publications that a-Toc failed to prevent photocarcinogenesis and even increased
protected skin from lipid peroxidation and oxidative the incidence of cell carcinoma.[11] With the aim of finding a
skin damage[3] and ultraviolet (UV)-induced damage[4] and better alternative to a-TAc, other pro-vitamins were synthe-
that it even inhibited the proliferation of UVB-induced sized with diverse moieties such as succinate,[11] amino-acid
tumours.[5,6] As endogenously existing a-Toc in the skin has esters[12] and phosphate,[13] and tocopherol glucoside.[14]
been established to be the first line of defence, exogenous Nevertheless, a-TAc still remains the derivative most com-
a-Toc has been included in many dermal products. monly used in cosmetic products.
However, a-Toc is unstable and is usually provided as its Essential fatty acids, such as linoleic acid (18:2n-6) and
prodrug ester, a-tocopherol acetate (a-TAc), which once gamma-linolenic acid (18:3n-6), form an important com-
permeated into the skin is supposed to be hydrolysed to its ponent of cell membranes and are therefore required for
active form.[7,8] The photoprotective benefits of topically both the structure and function of every cell in the body in
general, and for the skin’s integrity and health, in particular. the products have not been carried out, all fatty acid deriva-
Linoleic acid is the most abundant polyunsaturated fatty tives were routinely stored at -20°C until use.
acid (PUFA) in the skin epidermis. It exists in the tissue in
part as a moiety of the stratum corneum acylsphingolipids
NMR spectra
(acylceramides) involved in the epidermal barrier system.[15]
PUFAs of the w-3 type have been used for many years as a 1
H NMR spectra were recorded on a Bruker DMX-500
dietary supplement (mainly from fish oil) and have also operating at 500.1 MHz, and chemical shifts are reported in
been presumed to have beneficial effects in skin diseases. parts per million (d) using tetramethylsilane as the internal
Kew et al.[16] showed that w-3 PUFA can reduce markers of standard. Mass spectra were obtained using an Agilent
immune cell function. Clinical trials have shown that n-3 1100LC series (Waldbronn, Germany) and Bruker Esquire
PUFAs given to patients by the intravenous route have ben- 3000plus MS (Bremen, Germany) instruments. Electrospray
eficial and significant effects in psoriasis.[17] ionization-mass spectrometry-atmospheric pressure ioniza-
In this study we aimed to develop new pro-vitamins and tion electron spray interface, positive mode polarity meas-
compare their properties with those of a-TAc. As a-TAc urements were performed to obtain the [M + Na]+
possesses several drawbacks as described above, the use of molecular weights.
a combination of a-Toc and the essential fatty acids (n-6
and n-3) may be a better alternative than the current use
a-Tocopherol-palmitate
of a-TAc for topical treatment with vitamin E. A series of
a-Toc–fatty acid ester conjugates were synthesized and 1
H NMR (CDCl3): d 2.65 (t, 2H), 2.13 (s, 3H (CH3C)),
tested in vivo for skin permeation and bioconversion to 2.04 (s, 3H (CH3C)), 1.96 (s, 3H (CH3C)), 1.82 (m, 2H),
free a-Toc. It has been shown that well-selected ester con- 1.56 (m, 2H), 1.46 (m, 1H ((CH3)2CH)), 1.31 (br m, 28H),
jugates of fatty acids may replace a-TAc as they were 1.25 (m, 10H), 0.89 (m, 15H (CH3CH)). ESI-MS: m/z 692.1
found to be comparable with its cutaneous permeability
and metabolism.
a-Tocopherol-stearate
Table 1 Skin quantities of permeating a-Toc conjugates during 8 h after their cutaneous application to rats
Table 2 Skin quantities of totally detected free a-Toc as measured after cutaneous application of a-Toc–fatty acid conjugate to rats
NS, not significant. aThere was no statistical difference (Kruskal–Wallis test, P > 0.05) between the total a-Toc and the endogenous a-Toc in skin
treated with these two conjugates, suggesting that these compounds underwent essentially no hydrolytic degradation to free a-Toc in the skin.
separate group of untreated rats (control group). For every a-Toc linoleate, a-Toc linolenate and a-Toc palmitate
experimental set with a test group performed with a par- (P < 0.05, Kruskal–Wallis test), indicating skin metabolic
ticular conjugate, a control group was set in parallel. Skin activity. In contrast, the corresponding data obtained for
levels of the permeating derivatives ranged between 3 and a-Toc conjugates of the saturated fatty acid, a-Toc stearate,
10 nmol/cm2, 2–8 h after application. As shown in Figure 1, as well as for the conjugate of the unsaturated g-linolenate
the skin accumulation of a-TAc was somewhat higher than show no significant differences between the total and
the skin levels of the new conjugates; however, the skin endogenous a-Toc (P > 0.05; Table 2). Factors such as steric
levels of only a-Toc linoleate, a-Toc g-linolenate and a-Toc disturbances and reduced enzyme affinity may dictate
stearate were statistically different (P < 0.05, Kruskal–Wallis decreased rate of cleavage of these two conjugates. In a
test) compared with a-TAc levels, whereas the other conju- previous study,[22,23] we have shown that vitamin
gates penetrated into the skin to the same extent as a-TAc D3–polyunsaturated fatty acid conjugates quantitatively
(Table 1). The slightly increased permeability of a-TAc penetrated the skin and were metabolized therein. Unlike
compared with the fatty acid derivatives was apparently due the present study, we have shown that the g-linolenate con-
to its lower molecular weight (473 Da and 691–697 Da, jugate was metabolized by skin esterases to a free vitamin
respectively). The total levels of free a-Toc (i.e. the sum of D3, although to a lesser extent than a-linolenate conjugate.
the endogenous and the exogenous hydrolytic product) Due to these possible differences in the enzymatic hydroly-
were similar in all a-Toc-fatty acid conjugates and no sis between the various conjugates, further mechanistic
significant differences were found between the groups in research using purified esterases should be performed in
the total free a-Toc measured after a-TAc application at a separate in-vitro study to characterize the reaction
the various sampling times (Table 2, Figure 1; P < 0.05, limitations.
Kruskal–Wallis test). As shown by the differences between
the total free a-Toc and the endogenous a-Toc (Figure 1),
Multiple and extensive treatment of rats
the formation of free a-Toc by enzymatic hydrolysis of the
with a-TOC–fatty acid conjugates
conjugates occurred 2 h after application and this forma-
tion continued during the 8-h treatment. There were sig- To assess the skin penetration and biodegradation of a-Toc
nificant differences between the levels of total a-Toc and esters during chronic treatment of an extensive skin area,
endogenous a-Toc after application of a-TAc, a-Toc oleate, 4.25 mm solutions of a-TAc and a-Toc oleate were applied
(a) (b)
12 12
α-Toc linoleate
α-Toc oleate
Free α-Toc Free α-Toc
10 10
Endogenous α-Toc Endogenous α-Toc
Skin level (nmol/cm2)
6 6
4 4
2 2
0 0
2 4 6 8 2 4 6 8
Time (h) Time (h)
(c) (d)
α-Toc linoleate
12 12
Free α-Toc α-Toc γ-linoleate
Endogenous α-Toc Free α-Toc
10 10
Endogenous α-Toc
Skin level (nmol/cm2)
8 8
6 6
4 4
2 2
0 0
2 4 6 8 2 4 6 8
Time (h) Time (h)
(e)
12
α-TAc
10 Free α-Toc
Endogenous α-Toc
Skin level (nmol/cm2)
0
2 4 6 8
Time (h)
Figure 1 Skin accumulation of a-tocopherol (a-Toc) oleate (a), a-Toc linoleate (b), a-Toc a-linolenate (c) and a-Toc g-linolenate (d) in rats in com-
parison with a-tocopherol acetate (a-TAc) (e). Solutions (50 ml) of 4.25 mM vitamin E (as conjugates) in isopropyl palmitate were applied on the
dorsal skin of rats (1.7 cm2 surface area).
on rats’ backs (4 ¥ 4 cm; surface area of 16 cm2) once daily did occur after three daily applications to a similar extent
for three days. After the third application skin samples were found after single application (data not shown).
biopsied at 2, 4, 6 and 8 h and analysed. Unexpectedly, we
noted that the rats receiving a-TAc (but not a-Toc oleate)
Conclusions
showed signs of general weakness, nervousness, distress,
hypothermia and hypoxia. These symptoms started after We have shown that a-Toc–fatty acid conjugates penetrate
24 h and eventually caused death. The study of this group into the skin quantitatively to an extent comparable with
was halted and biopsies were taken from the corpses and the permeation of a-TAc. However, the bioconversion to
analysed. The data showed that a-TAc levels reached free a-Toc in the skin occurs only during treatment with
18–21 nmol/cm2 of skin surface area, more than twice the a-Toc oleate, a-Toc linoleate, a-Toc a-linolenate and a-Toc
levels found after a single application. Furthermore, no for- palmitate. Despite the common use of a-TAc in cosmetic
mation of free a-Toc was noted in the group treated with products and the claims that it is generally safe, we have
a-TAc. We have no explanation thus far for the phenom- found that a-TAc was lethal to rats treated with 1.15 mg/kg
enon in which a well-known and commonly used agent of this agent, which had been spread over 16 cm2 of skin
produces a toxic reaction upon extensive use. The dermal surface area for a long term. Similar treatment (once a day
lethal dose that kills 50% of rats of a-TAc is stated to be for 3 days) with a-Toc oleate did not harm the rats and no
> 3 g/kg for rats and the dose we used (2.4 mmol/ symptoms of fatigue, hypoxia or any other side effects were
kg = 1.15 mg/kg a-TAc) was well below this value so it noted. These findings suggest that a-Toc conjugates with
should not have caused any toxic effect. Zondlo Fiume[24] unsaturated fatty acids, such as linoleic, oleic and
reported that a-TAc is generally safe and results in no side a-linolenic acids, may be a good alternative as stable
effects; however, it was mentioned that in one animal test, vitamin E derivatives, rather than the commonly used but
a-TAc did produce sensitization. Also, in a double-blind questionable a-TAc ester.
study performed with 800 IU/day as a-TAc (= 800 mg
a-TAc/day) using eight men, two complained of severe Declarations
fatigue and weakness after 3 weeks. Both men had elevated
serum creatine kinase accompanied by creatinuria at 7 and Conflict of interest
14 days.[25] As noted above, a-Toc oleate did not produce The Author(s) declare(s) that they have no conflicts of
any side effects. The accumulation of a-Toc oleate in the interest to disclose.
skin was naturally elevated after three applications to values
of 6–9 nmol/cm2 compared with the values of 4–6 nmol/
Funding
cm2 obtained after a single application. In contrast to multi-
ple applications of a-TAc, in which bioconversion to a-Toc This research received no specific grant from any funding
did not occur, we have found that hydrolysis of a-Toc oleate agency in the public, commercial or not-for-profit sectors.
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