Synthesis and in vitro transdermal penetration enhancing activity of lactam N-acetic acid esters

Article abstract of DOI:10.1021/js950331n

A homologous series of N-acetic acid esters of 2-pyrrolidinone and 2- piperidinone has been prepared and evaluated for its ability to enhance the skin content and flux of hydrocortisone 21-acetate in hairless mouse skin in vitro. Enhancement ratios (ER) were determined for flux (J), 24-hour diffusion cell receptor cell concentrations (Q₂₄), and 24-h full-thickness mouse skin steroid content (SC) and compared to control values (no enhancer present). In addition, in an attempt to abrogate toxicity, these dermal penetration enhancers were designed to have the potential for biodegradation by dermal esterases. 2-Oxopyrrolidine-α-acetic acid dodecyl ester (5) showed the highest enhancement ratios for J (ER 67.33) and Q₂₄ (ER 180.66). 2- Oxopiperidine-α-acetic acid decyl ester (10) showed a high Q₂₄ (ER 162.07) but a lower J (ER 12.67). 2-Oxopyrrolidine-α-acetic acid decyl ester (3) showed the highest enhancement ratio for SC (ER 8.7). The ER Q₂₄ for 3, 5 and 10, as well as other lactam N-acetic acid esters in this work, were significantly higher than the ER found using Azone as enhancer.

Full text of DOI:10.1021/js950331n

Synthesis and in Vitro Transdermal Penetration Enhancing Activity of Lactam
N-Acetic Acid Esters
†
B
OZENA B. MICHNIAKX, MARK R. PLAYER
,
AND J. WALTER
S
OWELL, S .
R
Received August 7, 1995, from the Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina,
Columbia, SC 29208.
Final revised manuscript received November 14, 1995.
Accepted for publication November 22, 1995X.
Present Address: Section on Biomedical Chemistry, Laboratory of Medicinal Chemistry, National Institute of Diabetes, Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Abstract
0 A homologous series of N-acetic acid esters of 2-pyrrolidinone
and 2-piperidinone has been prepared and evaluated for its ability to
enhance the skin content and flux of hydrocortisone 21-acetate in hairless
mouse skin in vitro. Enhancement ratios (ER) were determined for flux
(J), 24-hour diffusion cell receptor cell concentrations (Q24), and 24-h
full-thickness mouse skin steroid content (SC) and compared to control
values (no enhancer present). In addition, in an attempt to abrogate
toxicity, these dermal penetration enhancers were designed to have the
Scheme 1s(a) BrCH2COBr, hexanes, 69 °C, 4 h. (b) (i) NaH, THF, room
potential for biodegradation by dermal esterases. 2-Oxopyrrolidine-
acetic acid dodecyl ester (5) showed the highest enhancement ratios for
J (ER 67.33) and Q24 (ER 180.66). 2-Oxopiperidine- -acetic acid decyl
ester (10) showed a high Q24 (ER 162.07) but a lower J (ER 12.67).
2-Oxopyrrolidine- -acetic acid decyl ester (3) showed the highest
R-
temperature, 4 h, (ii) THF, room temperature, 20 h.
R
Experimental Section
R
All chemicals were purchased from Aldrich Chemical Co. in the
highest available purity, except hydrocortisone 21-acetate, polyoxy-
ethylene 20 cetyl ether, and propylene glycol, which were obtained
from Sigma Chemical Co. Baxter Diagnostics, Inc., supplied reagent
grade solvents, except for methanol and acetonitrile, which were
HPLC grade. Azone was synthesized as previously described.24
Thin layer chromatography (TLC) was performed with Merck
precoated silica gel plates, type 60-F254, and visualization was
accomplished with iodine vapor. The melting points were determined
on an Electrothermal apparatus and are uncorrected. Infrared
spectra were recorded on a Beckman Acculab 4 spectrophotometer
either neat or using the potassium bromide technique when the
sample was a liquid or a solid at room temperature, respectively. 1H-
NMR spectra were obtained on a Bru¨ker AM 300 NMR spectrometer.
UV spectra were recorded on a Beckman DU-6 spectrophotometer.
Elemental analyses were conducted by Atlantic Microlabs, Atlanta,
GA, and were within (0.4% of theoretical for all compounds. All
compounds were purified by flash chromatography with 32-63 µm
silica gel obtained from Selecto, Inc., Kennesaw, GA.
enhancement ratio for SC (ER 8.7). The ER Q24 for 3, 5 and 10, as well
as other lactam N-acetic acid esters in this work, were significantly higher
than the ER found using Azone as enhancer.†
Penetration through the skin of topically administered
agents designed to have systemic activity has been a goal of
pharmaceutical scientists for many years. Numerous advan-
tages to topical drug administration exist, including ease of
access, improved patient compliance, absence of first-pass
metabolism, the ability to continuously administer potent
drugs or those with long half-lives, and the ability to deliver
agents which are unsuitable for oral administration.1
The major barrier to drug penetration is presented by the
uppermost layer of the skin, the stratum corneum. Many
methods have been used to decrease skin resistance to the
passage of drugs. These include the prodrug approach,2
iontophoresis,3 phonophoresis,4 and dermal penetration en-
hancers.5 This last approach is most convenient; however,
these compounds must be highly effective, show no irritancy
or toxicity, and be stable and pharmacologically inert.6 Many
groups of chemicals have shown enhancer activity, including
pyrrolidinones,7 urea and its derivatives,8 many surfactants,9
dimethyl sulfoxide and its derivatives,10 and Azone1 (lauro-
capram, 1-dodecylazacycloheptan-2-one). Azone has been the
most widely studied chemical penetration enhancer of the
1980s although a considerable number of analogues with
improved activity have been described.12-18
Several studies suggest that Azone acts by solubilizing or
increasing the entropy of highly ordered stratum corneum
lipids.19 Skin conductance experiments suggest that Azone
also increases skin water content.20 This is supported by the
fact that Azone is an effective enhancer for both hydrophilic
and lipophilic drugs.21
Azone analogues with the potential for biodegradability
have also been actively pursued.22,23 In an attempt to acheive
high flux-enhancing activity and potential biodegradibility,
we have incorporated an ester moiety into an Azone-like
structure.
Male hairless mice strain SKHl (hr/hr), 8 weeks old, were obtained
from Charles River Laboratories, Inc., Wilmington, MA.
Syn th esis of La cta m N-Acetic Acid Ester ssThe 5- and 6-mem-
bered lactams (1-14) were prepared with acetate esters at the
1-position (Table 1). The bromoacetate esters were prepared by
reaction of the appropriate alcohol with bromoacetyl bromide, and
this crude bromoacetate ester was used to alkylate the sodium salt
of the various lactams. This is a similar method to that used
previously to prepare hexahydro-2-oxo-1H-azepine-1-acetic acid alkyl
esters for evaluation as penetration enhancers25. Compounds 15 and
16, azepine homologues of the compounds prepared in the present
work, have been previously found to be essentially inactive as flux
enhancers.25 Empirical formulas, melting points (mp), and synthetic
yields are listed in Table 2.
Gen er a l P r oced u r e for Syn th esis of th e La cta m N-Acetic
Acid Ester ss2-Pyrrolidinone-1-acetic Acid Dodecyl Ester (5)sBro-
moacetyl bromide (21.19 g, 105 mmol) (CAUTION: lachrymator and
strong alkylating agent)26 was added to a solution of 1-dodecanol
(19.56 g, 105 mmol) in hexanes (250 mL). The solution was refluxed
for 4 h, after which time the solvent was removed in vacuo to yield
crude dodecyl bromoacetate. 2-Pyrrolidinone (0.85 g, 10 mmol) was
dissolved in Na-dried THF (100 mL) and NaH (0.24 g, 10 mmol)
(CAUTION: highly caustic, pyrophoric)26 was added. This mixture
was stirred for 2 h under a drying tube, during which time a cloudy
suspension formed. The crude dodecyl bromoacetate (3.07 g, 10 mmol)
in Na-dried THF (50 mL) was added dropwise over 10 min and the
mixture was stirred at room temperature for 20 h. The NaBr was
filtered off and the solvent removed in vacuo. The crude product was
X Abstract published in Advance ACS Abstracts, J anuary 1, 1996.
150 / Journal of Pharmaceutical Sciences
0022-3549/96/3185-0150$12.00/0
© 1996, American Chemical Society and
American Pharmaceutical Association
Vol. 85, No. 2, February 1996
+
+

Table 1
s
Ring Size and Alkyl Side Chain Length of Compounds 1
−16
Table 3
Content (SC) of Hydrocortisone and Hydrocortisone 21-Acetate (HC
HCA) for Compounds 1 16
sInitial Flux (J), 24-h Receptor Cell Concentration (Q24), and Skin
+
Compd
R
R′
−
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15a
16a
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)4
(CH2)4
(CH2)4
(CH2)4
(CH2)4
(CH2)4
(CH2)4
(CH2)5
(CH2)5
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
n-C8H17
n-C9H19
Compd (n
)
)
5)
8)
J (
µ
M/cm2 h)
Q24
(
µ
M)
SC (
µ
g/g)
Control (n
Azone
1
2
3
4
5
6
7
0.045 0.016
±
0.75
28.76
14.88
16.10
54.20
23.49
135.50
26.21
25.22
16.93
44.66
121.55
30.72
29.35
42.73
64.11
2.91
±
0.25
4.62
1.89
1.22
18.26
12.11
20.52
2.53
3.46
4.91
8.61
30.99
4.69
5.11
6.22
10.35
1.09
1.05
285.2
420.5
1054.3
1008.3
2471.5
1351.5
474.9
463.1
673.1
331.5
560.2
212.9
331.3
1277.7
1098.3
1755.0
449.2
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
21.6
36.9
40.9
n-C10H21
n-C11H23
n-C12H25
n-C13H27
n-C14H29
n-C8H17
n-C9H19
n-C10H21
n-C11H23
n-C12H25
n-C13H27
n-C14H29
n-C12H25
n-C14H29
0.88
0.85
0.79
1.72
0.69
3.03
0.81
1.16
0.83
0.60
0.57
1.64
1.07
0.54
1.70
0.05
0.09
±
0.25
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.27
0.19
0.22
0.15
0.84
0.30
0.21
0.20
0.11
0.22
0.15
0.03
0.15
0.19
0.02
0.01
312.5
816.2
100.2
108.4
110.0
100.0
125.5
163.2
90.9
112.1
454.9
275.1
563.4
39.8
8
9
10
11
12
13
14
15
16
a See ref 25.
Table 2s
Melting Point (if applicable), Yield, and Empirical Formulas of
Compounds 1−14
2.41
2167.4
85.9
Compd
Mp (
°
C)
Yield (%)
Formulaa
C18H35NO
Skin Retention StudiessAfter 24 h the skins were removed from
the diffusion cell and immersed in methanol several times for a total
of 5 s. The skins were then dried for 10 min, weighed, and cut up
with scissors, and 4 mL of methanol was added. The skin/methanol
was homogenized with a Kinematica, GmbH tissue homogenizer. The
samples were then filtered to remove debris, centrifuged if turbid,
and frozen at -80 °C until analysis. Recovery experiments showed
that skin steroid content and total corrected receptor concentrations
accounted for >95% of steroid administered.31
HPLC Analysis of SamplessSamples were analyzed by HPLC
utilizing a Waters 6000A solvent delivery system Model U6K injector
(injection volume 50 µL), a Waters differential UV detector Model
ALC 202, an Alltech C18 Versapack reverse-phase column (4.1 mm X
30 cm; 10 µm) at ambient temperature with a Hewlett Packard
integrator. Hydrocortisone and hydrocortisone 21-acetate were de-
tected at 254 nm and were eluted using a acetonitrile/water (4:6)
mobile phase at 1 mL/min.
Da ta An a lysissPenetration profiles for hydrocortisone were
constructed by plotting the receptor cell concentration of steroid (µM)
vs time (h). Profile analysis gave initial flux values, J (µM/cm2 h),
which were calculated from the slope of the linear region of the profile
(Figure 1, Table 3). In each case the flux graph, after a short lag
time, gave a linear portion consistent with steady-state Fickean
diffusion. Sink conditions were assumed to exist during the experi-
ments. Receptor cells contained only hydrocortisone (HC) whereas
skin samples contained a mixture of hydrocortisone 21-acetate (HCA)
and HC. Skin contents were calculated as micrograms of HCA + HC
per gram of hydrated full-thickness skin.
Azone
1
2
3
4
5
6
7
8
52
24
21
45
35
51
31
47
50
52
44
26
47
33
37
C14H25NO3
C15H27NO3
C16H29NO3
C17H31NO3
C18H33NO3
C19H35NO3
C20H37NO3
C15H27NO3
42.5−43.5
•
•
1/4H2O
1/4H2O
9
C
C
16H29NO3
17H31NO3
10
11
12
13
14
C18H33NO3
C19H35NO3
C20H37NO3
C21H39NO3
•
1/4H2O
28−
39−
38−
29
40
39
a All compounds were analyzed for C, H, and N and the results agreed to
0.4% of the theoretical values.
±
purified via flash chromatography with a hexanes/ethyl acetate (2:1)
to (1:2) eluotropic series to yield a clear oil (5) (1.58 g, 51%): TLC
(hexanes/ethyl acetate, 2:1) Rf ) 0.28; IR (neat) ν CdO 1720, 1622
cm-1 1H NMR (CDCl3) δ 0.83 (t, 3H, terminal CH3), 1.21 (m, 18H,
;
(CH2)9), 1.58 (m, 2H, CH2 â to O), 2.05 (m, 2H, 4-CH2), 2.32 (t, 2H,
3-CH2), 3.44 (t, 2H, 5-CH2), 4.00 (s, 2H, acetyl CH2), 4.07 (t, 2H, CH2
R to O) ppm. Anal. (C18H33NO3) Calcd: C, 69.41; H, 10.68; N, 4.50.
Found: C, 69.31; H, 10.65; N, 4.53.
P er m ea b ilit y
E xp er im en t ssIn
Vitro
Diffusion
Cell
ExperimentssHydrocortisone 21-acetate was chosen as the penetrant
(model drug). Full-thickness hairless mouse skins were placed in
unoccluded, modified Franz diffusion cells. The diffusional area was
3.14 cm2. The surface of the skin was maintained at a constant
temperature of 32 ( 0.5 °C. The receptor phase (volume 12 mL) was
continuously stirred at 600 rpm and consisted of isotonic phosphate
buffer (pH 7.4) with 0.1% v/v aqueous formaldehyde as preservative.27
The aqueous solubility of hydrocortisone 21-acetate is 0.28 mg/mL.28
In order to maintain sink conditions polyoxyethylene 20 cetyl ether
was added to the receptor phase as a solubilizer.29,30 Dorsal mouse
skin samples were placed in diffusion cells and were allowed to
equilibrate for 1.5 h. At this time the enhancer solution (0.4 M) in 5
µL propylene glycol was spread on the diffusional area. Although
solid enhancers are normally applied at their saturation solubilities
if they are under 0.4 M, all of the solid enhancers in the present work
(12-14) were soluble at 0.4 M and 32 ( 0.5 °C. One hour after
enhancer pretreatment, hydrocortisone 21-acetate (0.03 M) suspended
in 500 µL of propylene glycol was spread on the skins. Samples were
withdrawn from the receptor phase over 24 h. The receptor volume
was replenished with 100 µL of phosphate buffer after each sample.
Analysis of each sample was corrected for each previous sample which
had been removed. Each experiment was repeated five or more times.
The effectiveness of each compound was evaluated using enhance-
ment ratios (ER), where ER ) skin parameter from enhancer treated
skin/skin parameter from control (Table 4). Skin parameters used
were flux (J ), 24 h receptor concentrations of steroid (Q24), or total
steroid skin content (SC). Statistical treatment of the data consisted
of an analysis of variance (ANOVA) followed by a least significant
difference test (LSD) if the ANOVA indicated that a difference existed.
The level of significance (R) was selected to be 0.05.
Results and Discussion
Control experiments were run with only propylene glycol
and penetrant with no enhancer present. Permeation profiles
for controls, Azone, 5, and 10 are presented in Figure 1.
Control values were as follows: J ) 0.045 ( 0.016 µM/cm2 h,
Q24 ) 0.75 ( 0.25 µM, and SC ) 285.2 ( 21.6 µg/g for total
HC and HCA.
Azone produced a J ) 0.88 ( 0.25 µM/cm2 h, Q24 ) 28.76 (
4.62 µM, and SC ) 420.5 ( 36.9 µg/g for total HC and HCA.
Journal of Pharmaceutical Sciences / 151
Vol. 85, No. 2, February 1996
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Table 4sEnhancement Ratios for Flux (ERJ), 24-h Receptor Cell
(p < 0.05). As such, 3 or 16 may be useful in enhancing the
delivery of drugs which act locally within the skin, e.g. topical
methotrexate or 5-fluorouracil treatment for psoriasis.
Concentration (ERQ ), and Skin Content (ERSC) of Hydrocortisone and
24
Hydrocortisone 21-Acetate (HC
+
HCA) for Compounds 1−16
Another trend may be observed from consideration of ERQ
24
Compd
ERJ
ERQ
ERSC
24
values for 5, 7, 12, 14-16. That is, at least in this series,
smaller rings tend to cause transdermal enhancement to a
greater degree. For instance, when the side chain is an acetic
Control
Azone
1
2
3
4
5
6
7
1
1
1
19.55
18.89
17.55
38.22
15.33
67.33
18.00
25.78
18.44
13.33
12.67
36.44
23.78
12.00
37.77
1.13
38.35
19.84
21.47
72.27
31.32
180.66
34.94
33.63
22.57
59.55
162.07
41.02
39.13
56.97
85.48
3.87
1.5
3.7
3.5
8.7
4.7
1.7
1.6
2.4
1.2
2.0
0.8
1.2
4.5
3.9
6.2
1.6
7.6
acid dodecyl ester (5), this results in an ERQ of 180.66; 12
24
gives an ERQ of 39.13 and 15 gives an ERQ of only 3.87.
24
Also, compounds which give high ERSC’s give l2o4w ERQ ’s and
24
vice versa. For instance, 5 gives the highest ERQ , 180.66,
24
but only a low ERSC, 1.7. Conversely, 16 gives a high ERSC of
7.6 but an ERQ of only 3.26. Compound 14 is something of
an exception, a2s4 it has a high ERSC, 6.2, and ERQ , 85.48 (p
24
8
9
< 0.05 compared with control).
Since we were most interested in the possibility of trans-
dermal enhancement, we chose to prepare a homologous series
around the 5- and 6-membered ring lactams in hopes of
10
11
12
13
14
15
16
finding homologues with even higher ERQ ’s. To this end,
24
1-7 (2-oxopyrrolidine-R-acetic acid octyl to tetradecyl esters)
and 8-14 (2-oxopiperidine-R-acetic acid octyl to tetradecyl
esters) were synthesized.
1.96
3.26
The alkyl chain lengths for which ERQ was greatest were
24
observed to be different for the 2-oxopyrrolidine-R-acetic acid
ester group (1-7) and the 2-oxopiperidine-R-acetic acid ester
group (8-14). Q24 maxima were reached with acetic acid decyl
and tetradecyl ester moieties for 2-piperidinone (10 and 14);
in contrast, the 2-pyrrolidinone nucleus exhibited Q24 maxima
with acetic acid decyl and dodecyl ester substitutents (3 and
5).
It has been reported in the literature that even though some
activity may be observed with enhancers with very short alkyl
side chains or no side chain at all, the best enhancement
occurred when the length was about C8-C12. For example,
Sasaki et al. tested three pyrrolidinones, N-methyl-, N-hexyl-,
and N-lauryl-2-pyrrolidinone (LP), with 5-fluorouracil, triam-
cinolone acetonide, indomethacin, and flurbiprofen in vitro
using rat skin7. The N-hexyl derivative and LP were more
effective at enhancing the permeation of the hydrophilic drugs,
however LP was the most active of all three compounds tested.
We have also found that LP was an effective enhancer using
hairless mouse skin in vitro with hydrocortisone 21-acetate
as a model drug.24
Barry and Bennett have reported relatively high activity
using 2-pyrrolidinone and N-methyl-2-pyrrolidinone using
mannitol, progesterone, and hydrocortisone in vitro in human
skin.33 However, it must be borne in mind that each study
utilized a different animal model, vehicles, and experimental
protocols which may have contributed to the variation in data.
Our results with several pyrrolidinones, including 2-pyrroli-
dinone and N-methyl-2-pyrrolidinone, showed no activity in
hairless mouse skin in vitro using HCA and HC in propylene
glycol.24,34
Several efforts have been made to synthesize and investi-
gate biodegradable enhancers, such as dodecyl 2-(N,N-dim-
ethylamino)propionate and clofibric acid esters.31,35 The
degradation of the compounds would occur due to the presence
of esterases in mouse and human skins.32 This same rationale
was utilized in the present study; however, no attempts were
made to evaluate the extent of biodegradability of the en-
hancer tested.
The proposed mechanisms by which these compounds exert
their action have been investigated by several authors using
liposomal models, differential scanning calorimetry, small
angle X-ray diffraction, FT-infrared spectroscopy and other
techniques.36-39 Barry has proposed that the mechanism of
action may be explained using the lipid-protein-partitioning
(LPP) theory.40,41 This states that enhancers act by one of
three primary means. Enhancers may alter the lipid domain
Figure 1
s
Receptor phase steroid concentration (C) for control (
b), and with
Azone (
2
), 5 ( ) and 10 ( ) as permeation enhancer.
9
1
The receptor phase contained only HC, whereas skin samples
contained primarily HCA with some HC.
The lactam acetic acid esters (1-16) were developed as
novel enhancers which were not previously reported to show
transdermal or topical penetration enhancing activities. Ini-
tially, the acetic acid dodecyl and tetradecyl esters of ꢀ-ca-
prolactam25 (15, 16), 2-piperidinone (12, 14), and 2-pyrrolid-
inone (5, 7) were prepared.
With regard to the skin content enhancement of these
compounds a clear trend is noted. When the side chain
consists of an acetic acid tetradecyl ester, larger rings tend
to result in increased topical delivery of HCA; ERSC for 7 is
2.4, for 14, 6.2 and for 16, 7.6 (p < 0.05 compared with control).
This was despite the fact that the saturation solubility for 16
was only 0.14 M, a concentration three times lower than that
at which other lactam acetic acid esters were applied. Com-
pound 3, an exception to the above trend, gave the highest
ERSC of any enhancer in this work, 8.7 times over the control
152 / Journal of Pharmaceutical Sciences
Vol. 85, No. 2, February 1996
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+

of the stratum corneum, may interact with the protein
components, or may increase the partitioning of the model
drug, the coadministered vehicles, or water into the skin. The
alteration of the lipid domain occurs by fluidization of the
stratum corneum lipids.42-45
83 ( 11 for the pyrrolidinone analog, and 64 ( 6 for Azone.18
Taken together this indicates that enhancer length as well
as overall lipophilicity would be necessary descriptors for
predicting enhancer activity.
Molecular modeling studies have sugested a spoon shape
for Azone.56,57 The electrostatic fields around the ceramide
head groups have both electronegative and electropositive
regions on opposite sides of the molecules. The interaction
between these two areas within the molecules binds the head
groups together. Azone shows an electronegative site, at the
carbonyl function, but no positive site and it has been proposed
that intercalation of Azone into the ceramides will result in
an unbalanced electronegative site on the latter. This may
be the mechanism by which Azone exerts it permeation-
enhancing effects.55 This is supported by Williams, who
compared series of Azone analogs with either two electrone-
gative sites or both a positive and negative site. The former
compounds were enhancers whereas the latter actually slowed
drug permeation possibly due to the hypothesized interaction
with the ceramides.58 Some agents, however, increase skin
permeation rates by other mechanisms or their combination,
such as an influence on stratum corneum proteins, cosolvency,
or an alteration of the thermodynamic activity of the model
drug within the skin.5
Few investigators have studied the possible mechanism(s)
of action of pyrrolidinones and piperidinones; however, Azone
has been extensively studied. Recently, Bouwstra and Bodde
reviewed literature flux studies and subsequent differential
thermal analysis, small and wide angle X-ray diffraction,
freeze-fracture electron microscopy (FFEM), freeze-substitu-
tion electron microscopy (FSEM), and nuclear magnetic
resonance data and concluded that the mechanism of action
of N-alkyl-Azones on human stratum corneum is more com-
plex than previously thought.46 After evaluating 0.15 M
N-hexyl- through N-hexadecyl-Azones in propylene glycol, it
was determined that changes in stratum corneum barrier
properties depended on alkyl chain length. The influence on
stratum corneum of N-hexyl-Azone in propylene glycol was
similar to the effect produced by propylene glycol alone.
However, when the stratum corneum was pretreated with
N-octyl-Azone, small-angle X-ray diffraction studies revealed
lipid fluidization. Both FFEM and FSEM confirmed these
findings and showed that the fluidization was occurring to a
greater extent in the center of the intercellular space rather
than close to the cell boundaries. 1H-NMR experiments also
revealed that N-dodecyl-Azone induced a “liquid microenvi-
ronment” within the stratum corneum.
These findings may be different in other animal skins such
as the hairless mouse and will depend on the vehicle and the
model drug used. The effect of model drug changes and skin
type is well-illustrated by Hadgraft et al.47 Schuckler and Lee
in a recent study found that the extent on the N-dodecyl-Azone
accumulation in human stratum corneum could be correlated
with its effect on the diffusion coefficient of diazepam. This
coefficient was highest when the skin content of the enhancer
was 12% w/w. This high quantity was surprising and implied
that on a molar basis there was more Azone present in the
stratum corneum than lipid.48
Studies on model systems such as skin lipids and phospho-
lipids have shown that N-dodecyl-Azone has an effect on the
hydrocarbon chains inside the bilayer structures.49,50 How-
ever, when lamellar phases were prepared from brain cera-
mides, free fatty acids, and cholesterol or from hydrated
phosphatidylcholine, no fluidization of lipids was observed.51,52
It has been shown in the literature that there is a parabolic
effect of alkyl chain lengths of enhancers, with C10-C12 being
the most active.53 Pyrrolidinone enhancers have also been
shown to have higher activity with a N-dodecyl side chain,
compared to N-methyl or N-hexyl.7,54 It may be of interest to
note that this chain length corresponds to the length of the
steroid nucleus of cholesterol, suggesting that the mechanism
of action of these agents involves the disruption of ceramide-
cholesterol or cholesterol-cholesterol interactions.55
In summary, the present study examined a homologous
series of N-acetic acid esters of 2-pyrrolidinone and 2-piperi-
dinone. The highest flux-enhancing activity was noted for
2-oxopyrrolidine-R-acetic acid dodecyl ester (5), which pro-
duced a 70-fold increase in flux of HCA over control values (J
) 3.03 ( 0.84 µM/cm2 h with 5; J ) 0.045 ( 0.016 µM/cm2
h
with control); Q24 values were also high, 135.50 ( 20.52 µM
for 5, and 0.75 ( 0.25 µM for control (p < 0.05). Azone gave
values of 0.88 ( 0.25 µM/cm2 h for flux and 28.76 ( 4.62 µM
for Q24 (p < 0.05). Skin content of steroid for 5 was similar
to that of Azone (474.9 ( 108.4 µg/g for 5; 420.5 ( 36.9 µg/g
for Azone). The highest skin steroid content was observed
for 2-oxopyrrolidine-R-acetic acid decyl ester (3): 2471.5 (
816.2 µg/g (p < 0.05).
The highest Q24 for the 2-oxopiperidinone-R-acetic acid
esters was observed for the decyl ester (10), 121.55 ( 30.99
µM, while the highest skin content was recorded for the
tetradecyl ester (14), 1755.0 ( 563.4 µg/g (p < 0.05). As a
number of these compounds are significantly more active than
Azone, they have potential for further development as effective
dermal penetration enhancers.
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Acknowledgments
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The authors would like to thank Hoffmann-La Roche, Inc., for their
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J S950331N
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