Synthesis and Enhancing Effect of Transkarbam 12 on the Transdermal Delivery
of Theophylline, Clotrimazole, Flobufen, and Griseofulvin
Alexandr Hraba ´lek,1,2,4Pavel Dolez ˇal,3Kater ˇina Va ´vrova ´,1,2Jarmila Zbytovska ´,3Toma ´s ˇ Holas,1
Jana Klimentova ´,1and Jakub Novotny ´1
Received October 11, 2005; accepted December 21, 2005
Purpose. Dodecyl-6-aminohexanoate (DDEAC) is a transdermal permeation enhancer with excellent
activity, low toxicity, and no dermal irritation. We hypothesized that DDEAC reacts with air CO2to
form a two-chain ammonium carbamateVTranskarbam 12 (T12)Vwhich is responsible for the
Methods. DDEAC and T12 were synthesized, their structures were confirmed by spectral methods, and
their enhancing activity was studied using the Franz diffusion cell and human skin. A high-performance
liquid chromatography method was developed for determination of T12, and its biodegradability was
evaluated using porcine esterase.
Results. Only the carbamate salt T12 was responsible for the high enhancing activity; DDEAC tested
under argon to avoid reaction with CO2was inactive. T12 enhanced transdermal permeation of drugs
covering a wide range of physicochemical properties, including theophylline (enhancement ratio up to
55.6), clotrimazole (7.7), flobufen (5.0), and griseofulvin (24). The activity was pH-dependent, further
confirming the importance of the carbamate structure. The metabolization of T12 followed a second-
order kinetics with t1/2= 31 min.
Conclusion. Our results indicate that T12 is a promising biodegradable permeation enhancer for a wide
range of drugs, and the structurally novel group of carbamate enhancers warrants further investigation.
KEY WORDS: ammonium carbamate; biodegradability; permeation enhancer; transdermal drug
Transdermal drug delivery offers numerous advantages
over the conventional routes of administration; however,
poor permeation of most drugs across the skin barrier
constitutes a serious limitation of this methodology. One of
the approaches used to enlarge the number of transdermally
applicable drugs uses permeation enhancers. These com-
pounds promote drug permeation through the skin by a
reversible decrease of the barrier resistance (1).
1-Dodecylazepan-2-one (Azone) was the first molecule,
specifically designed as a skin permeation enhancer (Fig. 1)
(2). This compound enhances the skin transport of a wide
variety of drugs (for reviews on Azone, see (3,4)) and serves
as a lead compound for structural modifications (5Y9). The
structureYactivity relationships of amphiphilic permeation
enhancers have been reviewed recently (10).
We have previously reported the permeation-enhancing
activity of a series of 6-aminohexanoic acid esters (11,12). The
compounds were designed as acyclic Azone analogs (Fig. 1),
the flexible structure of which could adopt an optimum
conformation, and thus interact more readily with the
stratum corneum components. Moreover, the ester linkage
offers the possibility of degradation by skin esterases into
nontoxic metabolites. These compounds displayed excellent
enhancement activities for theophylline, approximately an
order of magnitude higher than that of AzoneVenhancement
ratios of dodecyl-6-aminohexanoate (DDEAC) and Azone
dispersed in water were 35.0 and 5.4, respectively, and 16.7
and 1.1, respectively, when applied in olive oil. The acute
toxicity of DDEAC after intraperitoneal administration to
mice was lower than that of Azone [LD50of DDEAC and
Azone were 352 mg/kg (11) and 232 mg/kg (13), respective-
ly], and the compound did not exhibit any acute dermal
irritation in vivo on rabbits (11).
Recently, we have found that the behavior of the
enhancer does not correspond to that of an amino ester
molecule, with the sample, moreover, containing only 91% of
DDEAC. This finding could be explained by the formation
0724-8741/06/0500-0912/0#2006 Springer Science + Business Media, Inc.
Pharmaceutical Research, Vol. 23, No. 5, May 2006 (#2006)
1Department of Inorganic and Organic Chemistry, Faculty of
Pharmacy, Charles University in Prague, Heyrovske ´ho 1203, 50012
Hradec Kra ´love ´, Czech Republic.
2Centre for New Antivirals and Antineoplastics, Faculty of Pharma-
cy, Charles University in Prague, Heyrovske ´ho 1203, 50012 Hradec
Kra ´love ´, Czech Republic.
3Department of Pharmaceutical Technology, Faculty of Pharmacy,
Charles University in Prague, Heyrovske ´ho 1203, 50012 Hradec
Kra ´love ´, Czech Republic.
4To whom correspondece should be addressed. (e-mail: Alexandr.
of an ammonium carbamate salt by the trapping of carbon
dioxide by the free amino group. This two-chain enhancer
with a carbamate salt forming its polar head, termed Trans-
karbam 12 (T12, Fig. 1), has been suggested to be responsible
for the enhancing effect (14,15).
The purpose of this study was to confirm the structure of
this promising enhancer and evaluate its activity in a greater
detail. We aimed at preparing both possible structures,
DDEAC and T12, in pure form, and directly comparing
their enhancing activities to find the contribution of both
structures to the permeation-enhancing effect. In addition,
we have evaluated the enhancing activity of T12 toward a
broader spectrum of drugs, a preliminary in vitro biodegrad-
ability using porcine esterase, and developed a high-perfor-
mance liquid chromatography (HPLC) method for the
determination of this substance.
MATERIALS AND METHODS
Chemicals and Instrumentation
All chemicals, HPLC solvents, theophylline, clotrima-
zole, griseofulvin, and esterase [from porcine liver, suspen-
sion in 3.2 M (NH4)2SO4, pH 8, 250 U/mg protein, 15 mg
protein/ml] were purchased from Sigma-Aldrich (Schnell-
dorf, Germany). Flobufen (16) was obtained from VUFB
(Prague, Czech Rep.). HPLC columns (see below) were
purchased from Merck (Darmstadt, Germany).
Infrared (IR) spectra were recorded on a Nicolet Impact
400 apparatus equipped with a DTGS detector with a reso-
lution of 4 cmj1.
(NMR) spectra were measured on a Varian Mercury-Vx BB
300 instrument, operating at 300 MHz for1H and 75 MHz
for13C. Fast atom bombardment (FAB) mass spectra were
run on a ZAB-2SEQ mass spectrometer using a cesium ion
gun at 25 kV. The sample was dissolved in toluene, and
1 ml of the solution was added to the matrix. For +FAB,
2-nitrophenyl(octyl)ether was used as a matrix and
diethanolamine for jFAB. Elemental analysis (C, H, N)
was performed on a Fisons EA 1110 CHNS-O elemental
analyzer. The melting point was measured on a Kofler
apparatus and is uncorrected.
13C nuclear magnetic resonance
Dodecyl-6-aminohexanoate. DDEAC was synthesized as
described previously (11). To obtain pure amino ester without
traces of a carbamate, the substance was heated at reflux in
CHCl3for 5 min and was then dried using a stream of argon.
The process was repeated twice, and the product was stored
under argon to avoid interaction with carbon dioxide. MW =
299.5 g/mol. Colorless oil. Yield: 89%. IR (CHCl3): nmax3448,
3375, 2928, 2856, 1725, 1467 cmj1;
CDCl3): d 4.04 (2H; t; J = 6.6 Hz, OCH2); 2.69 (2H; t; J = 6.9
Hz, NCH2); 2.29 (2H; t; J = 7.5 Hz, COCH2); 1.86 (2H; bs,
NH2); 1.70Y1.55 (4H; m; 2CH2); 1.55Y1.40 (2H; m; CH2);
1.40Y1.15 (20H; m; 10CH2); 0.87 (3H; t; J = 6.9 Hz; CH3);
13C NMR (75 MHz, CDCl3): d 173.80; 64.46; 41.82; 34.25;
33.04; 31.88; 29.60; 29.55; 29.49; 29.32; 29.22; 28.60; 26.37;
25.89; 24.76; 22.66; 14.10.
ycarbonyl)pentylcarbamate (T12). Method A: Neat DDEAC
was placed into a CO2 atmosphere for 2 h. The yellowish
crystalline product was recrystallized from toluene (at dissolv-
ing T12 in toluene, the temperature cannot exceed 50-C!).
Method B: CO2was introduced into an ethereal solution of
DDEAC for 0.5 h. The crystalline product was filtered,
washed with diethylether, and dried in vacuo at room
temperature. MW = 643.0 g/mol. Yield: 85% (A)Y89% (B).
White crystals, mp = 62Y65-C. IR (KBr): nmax3360, 1734,
1735, 1650, 1617 cmj1;1H NMR (300 MHz, 70 mg of T12/0.7
ml CDCl3, 5 drops of dry pyridine): d 7.09 (3H; bs; NH3
(1H; bs; NH); 3.95 (4H; t; J = 6.8 Hz; 2OCH2); 3.05Y2.90 (2H;
m; CH2NHCOO); 2.80Y2.55 (2H; m; CH2NH3
J = 7.4 Hz; 2CH2CO); 1.60Y1.05 (52H; m; 26CH2); 0.78 (6H; t;
J = 6.6 Hz; 2CH3);13C NMR (75 MHz, 70 mg of T12/0.7 ml
CDCl3, 5 drops of dry pyridine): d 173.53 (COO); 161.98
(NHCOOj); 64.20; 41.02; 40.50; 34.06; 31.71; 30.04; 29.43;
29.38; 29.33; 29.14; 29.06; 28.44; 26.27; 25.72; 24.55; 22.47;
13.89 ppm; jFAB: m/z 342.2 [RNHCOO]j, 298.2 [RNH]j;
+FAB: m/z 300.2 [RNH3]+[R = C12H25OOC(CH2)5]; Anal.
(C37H74N2O4) C, H, N.
1H NMR (300 MHz,
+); 2.20 (4H; t;
Human cadaver skin was purchased from the Tissue
Bank of the Teaching Hospital in Hradec Kra ´love ´, Czech
Republic. The skin grafts from identical areas on a thigh from
donors of either sex (60% female; with mean age 66 T 9
years) were collected under aseptic conditions using a
dermatome to gain samples of a thickness of about 300 mm.
The skin samples were maintained in a wet chamber, and
then adjusted into polyethylene bags, which were evacuated,
closed, and stored at j20-C.
The permeation-enhancing activities of T12 and
DDEAC were evaluated in vitro using the Franz diffusion
cells (17). The skin samples were slowly thawed (at 4-C first
and then at ambient temperature), cut into pieces, and
mounted into the cells dermal side down to leave a diffusion
area of 2 cm2. Closely adjacent skin pieces cut from each
graft were used for the permeations with the control samples
Fig. 1. Azone, its acyclic analog dodecyl-6-aminohexanoate
(DDEAC), and the corresponding alkylammoniumYalkylcarbamate
Transkarbam 12 (T12).
913Transdermal Permeation Enhancer Transkarbam 12
and the pertinent enhancer. The acceptor compartment of
the cell was filled with the pertinent acceptor phase (Table I)
and allowed to equilibrate in a 32-C water bath with stirring
for 30 min. The precise volume of the acceptor compartment
(16Y18 ml) was measured for each cell and was included into
the calculations. The donor sample of 750-ml volume was
applied on the skin surface, and the donor compartment of
the cell was occluded with a cover glass. Samples of the
acceptor phase of 0.6-ml volume were withdrawn at
predetermined intervals over 48 h and were replaced with
fresh acceptor phase.
The donor samples were prepared by dispersing the
pertinent drug in a given vehicle (see Table I for details). The
samples with the enhancers were prepared by first dispersing
the enhancer (1%) in the vehicle and then adding the drug.
At this concentration, T12 was suspended, with partial
dissolution. The suspensions were stirred for 5 min at 45-C
(except for griseofulvin, which was dispersed at room
temperature), allowed to equilibrate at 37-C for 24 h, and
redispersed before application on the skin.
To evaluate the effect of DDEAC in the form of a pure
amino ester, the substance was dissolved in CHCl3to cleave
the carbamate salt that could have been formed during
handling, and the solvent was evaporated under a stream of
argon. A vehicle flushed with argon was added to the liquid
enhancer, and the mixture was stirred at 45-C for 5 min.
Theophylline was subsequently dispersed, and a stream of
argon was bubbled through the mixture for several minutes to
exclude the presence of carbon dioxide. The donor compart-
ment of the cell was prefilled with argon and, after the
application of the DDEAC-containing sample, covered to
prevent the reaction with air carbon dioxide and the formation
of the carbamate salt during the permeation experiment.
All donor samples were realized to be saturated because
they were partly overloaded by the drug as well as the
enhancer at the given conditions. The addition of the
enhancers very probably had an effect on the solubility of
drug and vice versa, but thermodynamic activity of both of
them within the donor samples was realized to be maximal
under the whole permeation experiment. The reported pH
values were adjusted under a pH-metric control and checked
prior the application, so the pH values of the samples applied
to the skin were the same for both the enhancer-containing
and control samples.
HPLC Determination of the Model Drugs
High-performance liquid chromatography analyses were
performed using an ECOM LCP high-pressure pump,
ECOM autosampler, Spectra Physics 8440 UV detector, and
CSW 1.7 integrating software (Prague, Czech Republic). The
samples of the acceptor phase were injected into the column
without further treatment.
The amount of theophylline in the acceptor phase
samples was determined using a LiChroCART 250-4
column (LiChrospher 100, RP 18, 5 mm) and methanol/0.1
M NaH2PO44:6 v/v as the mobile phase at a flow rate of 1.2
ml/min. The detector wavelength was set at 272 nm. The
retention time of theophylline was 3.3 min.
Clotrimazole was determined using a LiChroCART 250-
4 column (LiChrospher 60 RP-Select B, 5 mm) and methanol/
0.025 M K2HPO45:1 v/v as the mobile phase at a flow rate of
1.2 ml/min. The effluent was monitored at 227 nm, and the
retention time of clotrimazole was 4.2 min.
Flobufen was determined using a LiChroCART 125-4
column (LiChrospher 100, RP 18, 5 mm) and acetonitrile/
phosphate buffer at pH 3.0, 6:4 v/v, as the mobile phase at a
flow rate of 1.2 ml/min. The effluent was monitored at 279
nm, and the retention time of flobufen was 2.5 min.
Griseofulvin was determined using a LiChroCART 125-
4 column (LiChrospher 100, RP 18, 5 mm) and methanol/
water 7:3 v/v as the mobile phase at a flow rate of 1.0 ml/min.
The effluent was monitored at 291 nm, and the retention time
of griseofulvin was 2.3 min.
Thecumulative amountof the drughaving penetrated the
skin, corrected for the acceptor sample replacement, was
plotted against time. The steady-state flux (mg/cm2/h) was
calculated from the linear region of the plot. The enhance-
Table I. The Properties of the Model Drugs and the Composition of the Donor Samples and Acceptor Phases Used
for the Permeation Experiments
Model drugTheophyllineClotrimazoleFlobufen Griseofulvin
Drug amount (%)
PBS 7.2/PG/E 5:4:1
PBS 6.6/PG 1:1
Acceptor phase PBS 7.4/E 6:4
w = water; t7.3 = Tris buffer at pH 7.3, etc.; PG = propylene glycol; E = ethanol; PBS = phosphate-buffered saline with 0.03% NaN3.
aChemical Abstracts; calculated by Advanced Chemistry Development (ACD/Labs) Software Solaris V4.67.
bThe Merck Index, Merck & Co., Inc., Whitehouse Station, NJ, USA.
cSee Kuchar et al. (16).
914 Hraba ´lek et al.
ment ratio (ER) value was calculated as the ratio of the flux
of the drug with an enhancer and the flux of the drug alone.
The data are presented as means T SD obtained using
the skin samples from at least three donors. The statistical
significance of the differences between the drug permeation
withand without the enhancer was analyzed using the Student’s
t test. A value of p < 0.05 was considered significant.
Freshly boiled water of 9.8-ml volume with 100 ml of
0.2% (w/v) T12 solution in acetonitrile (i.e., 200 mg of T12)
was stirred in a water bath at 37-C for 10 min. Subsequently,
100 ml of a 1:1000 dispersion of porcine esterase in water (0.4
U of the enzyme) was added. At predetermined time
intervals, 5 ml of acetonitrile was added to the reaction
vessel to quench the hydrolysis. The mixture was carefully
evaporated at 35-C and dried in vacuo over P2O5. The same
reaction was carried out without addition of the enzyme.
The residue was dissolved in 5 ml of chloroform, and 20
mg of 3,5-dinitrobenzoyl chloride (DNBC) and 0.2 ml of
triethylamine were added. The mixture was heated at reflux
for 0.5 h, evaporated to dryness, and allowed to stand over
H2SO4 in vacuo overnight to remove the excess triethyl-
amine. The derivatized mixture was then dissolved in 1.0 ml
of acetonitrile and analyzed by HPLC using a LiChroCART
125-4 column (LiChrospher 100, RP 18, 5 mm, Merck). The
mobile phase consisted of a mixture of acetonitrile/water/
acetic acid, 80:20:1, and a flow rate of 1.5 ml/min was used.
DNBYT12 solution of 10-ml volume was injected onto the
column and monitored at 230 nm, with the retention time of
9.3 T 0.4 min. The calibration provided good linearity over
the concentration range of 0.05Y200 mg/ml.
The presence of dodecanol on the reaction was deter-
mined by thin-layer chromatography (TLC). The reaction
mixture after 180 min was evaporated to dryness and
analyzed on TLC plates (silica gel 60 F254, aluminum back,
Merck), using chloroform/methanol 9:1 mobile phase and
standard T12 and dodecanol solutions. The spots were visual-
ized by Ce(SO4)2, H3[P(Mo3O10)4], and H2SO4at 180-C.
Synthesis of DDEAC and T12
Initially, DDEAC was synthesized, as described in our
preceding article (11). IR spectra of the product measured in
a KBr tablet showed that the substance contained, at least in
part, an ammonium carbamate salt. Thus, the process
formerly described as crystallization was actually a reaction
of the amino ester DDEAC with carbon dioxide, yielding the
crystalline ammonium carbamate salt T12. Therefore, the
term DDEAC was previously used incorrectly, and, in this
article, it is used purely for the amino ester, whereas the
carbamate is termed T12.
To confirm the structure of T12 and to compare its effect
with DDEAC, both substances were prepared. Because of
high affinity of DDEAC to carbon dioxide, it was not
possible to isolate the pure amino ester using the previously
described procedure (11). Moreover, a relatively fast intra-
molecular cyclization of DDEAC to (-caprolactam was
observed. Therefore, it was more convenient to prepare
DDEAC by the degradation of the carbamate immediately
before permeation experiments. Pure T12 can be prepared
either by reaction of the liquid amino ester with CO2or by
introducing CO2into its ethereal solution. The latter method
yields an easy-to-isolate product of high purity.
Confirmation of the Structure of T12
Infrared spectra of T12 in a KBr tablet showed a strong
carbamate carbonyl stretching vibration at 1617 cmj1with a
shoulder at 1650 cmj1, and a doublet of ester carbonyl
vibration at 1735 and 1743 cmj1, indicating two ester groups
with different hydrogen bonding. Similar results were ob-
tained in Nujol suspension and using attenuated total reflec-
tion crystal. However, when recorded in a CHCl3solution, no
such vibration was observed. For a detailed IR and Raman
spectroscopic study of T12, see Zbytovska ´ et al. (15).
For the NMR spectroscopy, a suitable solvent that
dissolves but not decomposes the carbamate salt was needed.
Because of a very low solubility of T12 in most organic
solvents, the NMR spectra were recorded in CDCl3saturated
with dry pyridine with high concentration of the sample (10%
w/v). The13C NMR spectrum showed a signal at 161.98 ppm,
attributed to the carbonyl carbon of the carbamate salt, and
two resonances at 41.02 and 40.50 ppm, corresponding to the
methylene carbons next to NH3
structure; two signals of the methylene hydrogens adjacent to
ammonium and carbamate nitrogen were present at the ratio
FAB was used for the mass spectrometric characteriza-
tion of T12. Positive FAB showed an ion at m/z 300.2, which
corresponds to the ammonium salt C12H25OOC(CH2)5NH3
In the negative FAB spectrum, the compound showed a
strong m/z 342.2 ion, corresponding to the carbamate anion
C12H25OOC(CH2)5NHCOOjtogether with a weaker ion at
m/z 298.2 (loss of CO2). Furthermore, CHN analysis results
were in accordance with the carbamate structure.
+and NHCOOj, respectively.
1H-NMR spectrum further confirmed the carbamate
Flux and ER values for the permeations of theophylline,
clotrimazole, flobufen, and griseofulvin are summarized in
Table II. Typical permeation profiles for each drug are shown
in Fig. 2. Theophylline flux from both buffers and non-
buffered aqueous vehicle was approximately 3.5 mg/cm2/h.
While T12 exhibited high enhancing activity for theophylline
from all tested donor vehicles (with fluxes of approximately
170 mg/cm2/h, and the corresponding ER values up to 55.6),
DDEAC was completely inactive.
The data in Table IIb show clotrimazole permeation
from vehicles at different pH in the presence of T12. The flux
values slightly increased with increasing pH of the donor
sample up to 1.0 mg/cm2/h. The addition of 1% of T12
increased the permeation 7.0- and 7.7-fold at pH 7.3 and 8.3,
respectively; however, it had a very little effect at pH 9.3.
The data for flobufen permeation are listed in Table IIc.
The flux values without an enhancer were approximately 1
mg/cm2/h; the addition of T12 led to flux values of 4.6, 4.0,
and 2.9 mg/cm2/h at pH 7.0, 8.0, and 8.7, respectively.
915 Transdermal Permeation Enhancer Transkarbam 12
Flux and ER values for griseofulvin permeation are
shown in Table IId. In two control experiments, no griseo-
fulvin was detected in the acceptor samples, and these
experiments were excluded from the calculation of the mean
flux and, consequently, the ER value and t tests. Therefore,
the real flux is likely to be lower and the ER value higher.
The addition of 1% of T12 to the donor sample, however,
resulted in a reproducible flux value of 4.1 mg/cm2/h.
Enzymatic Hydrolysis of T12
Figure 3 shows a plot of T12 concentration [T12] vs. time
in the presence of porcine esterase. The degradation of the
ester bond followed a second-order kinetics with the
apparent second-order rate constant k = 0.0175 molj1m3sj1
and the estimated half-life t1/2having been approximately 31
min. No decrease in T12 concentration was observed in the
same experiment without addition of the esterase, which
excludes chemical hydrolysis. For the determination of T12, a
reversed-phase HPLC method was developed. T12 was
converted into DDEAC and derivatized by DNBC for
Table II. Permeation Characteristics of the Model Drugs and the
Enhancing Effect of 1% DDEAC and T12, Respectively
Vehicle/enhancerFlux T SD (mg/cm2/h)ER
3.8 T 1.0
166.6 T 34.0a
3.6 T 0.7
3.5 T 1.9
172.2 T 75.4a
4.9 T 1.9
3.2 T 1.3
176.7 T 78.7a
4.2 T 0.6
0.5 T 0.1
3.3 T 0.4a
0.6 T 0.1
3.9 T 0.8a
1.0 T 0.3
1.4 T 0.3
0.9 T 0.2
4.6 T 1.0a
1.1 T 0.4
4.1 T 1.2a
1.5 T 0.5
2.9 T 1.1
4.1 T 0.8a
n = 4Y8
w = water; t7.3 = Tris buffer at pH 7.3, etc.; PG = propyleneglycol; E =
ethanol; DDEAC = dodecyl-6-aminohexanoate; T12 = Transkarbam
12; ER = enhancement ratio.
aSignificantly different from control (p < 0.05).
bIn two control experiments, no griseofulvin was detected in the
acceptor samples. These data were excluded from the calculation of
the mean flux; consequently, the actual value is lower and the
corresponding ER is higher.
Fig. 2. Examples of the permeation profiles of the model drugs through the human skin from a control sample (&), and in the presence of 1%
T12 ()) and 1% DDEAC (Ì), respectively. Drug, vehicle: (A) theophylline, w, (B) clotrimazole, t7.3/PG/E, (C) flobufen, t7.0/PG, (D)
Fig. 3. In vitro hydrolysis of T12 in the presence of porcine esterase
(second-order reaction). T12 concentration [T12] = j4.9 + 16.2 ?
ejt/9.4+ 19.0 ? ejt0/244; R2= 0.99988. Insert: plot of 1/[T12] against
time, p < 0.0001; k = 0.0175 molj1m3sj1; t1/2= 31 min.
916 Hraba ´lek et al.
ultraviolet detection. The detection limit was 50 ng/ml. As this
method describes only decomposition of T12 and not the
appearance of metabolites, the presence of the dodecanol has
been confirmed by TLC (chloroform/methanol 9:1, Rf values
for dodecanol and T12 were 0.7 and 0.1, respectively).
Skin permeation enhancer DDEAC was designed as an
acyclic Azone analog. Because IR and NMR spectra in
chloroform confirmed the amino ester structure in the
previous study, we believed that opening of the azepan-2-
one cycle of Azone and substitution of the amide with an
ester group were the reasons of the markedly increased
activity of this novel enhancer (11). However, this substance
showed a crystalline structure with relatively high melting
point and was insoluble in a number of organic solvents,
which was unusual of the suggested amino ester structure.
Moreover, an acidimetric titration showed that the substance
contained only 91% of DDEAC.
Thus, it was suggested that the structure of the
enhancer is actually an alkylammonium salt of an alkylcar-
bamic acidVT12Vproduced by the reaction of DDEAC
with carbon dioxide. The formation of carbamates from
ammonia and primary and secondary amines is a well-known
reaction of great importance in biology and industrial
applications. For an extensive review on carbamate deriva-
tives, see Dell’Amico et al. (18). A similar carbamate struc-
ture was found in skin permeation enhancers, based on
tranexamic acid (trans-4-aminomethylcyclohexanecarboxylic
acid) derivatives (19).
In this study, the carbamate structure of the enhancer
was fully confirmed by spectral methods. Therefore, the
activity and toxicity data reported previously apply to the
carbamate salt, i.e., T12 (11). The reason why the IR and
NMR spectra confirmed the amino ester structure in the
previous study is the reversibility of the reaction. T12 can
decompose into DDEAC and carbon dioxide in an acidic
environment or upon heating (15). It was found during this
work that trace amounts of protons in chloroform were able
to decompose the carbamate; therefore, the previously
measured spectra were consistent with the structure of
DDEAC. This chloroform-mediated carbamate cleavage
was used in the preparation of pure DDEAC.
Previously investigated thermotropic behavior of T12
showed the first transition at about 53-C, related to a change
in the carbamate polar head (15). This explains why CHN
analysis had confirmed DDEAC structure (11), as the sample
for the analysis was dried in vacuo at 54-C. Although CO2
stays connected to the molecule via a noncovalent interaction
and is not being released during this transition (15), the
vacuum could shift the equilibrium toward DDEAC by
removing the loosely bound CO2. The analysis of a sample,
dried at ambient temperature, was in accordance with the
structure of T12.
As already noted, T12 can be decomposed into DDEAC
in a slightly acidic environment. As stratum corneum, the site
of action of permeation enhancers, is of acidic nature, the
decomposition of T12 into DDEAC could be expected.
Therefore, we aimed at identifying the nature of the actual
active substance, i.e., either T12 or the released DDEAC.
The enhancement activities of T12 and DDEAC were
compared using the Franz diffusion cell, human skin, and
theophylline as a model drug, i.e., under the same conditions
as in the previous study (11). Theophylline is a weak base of
medium lipophilicity (log P õ 0) and a small molecular
weight (Table I). The activity of DDEAC was evaluated
under argon, including sample preparation, to avoid its
reaction with carbon dioxide. This enabled us to compare
directly the enhancement ability of the free amino ester
DDEAC and its corresponding ammonium carbamate salt
T12. The results demonstrated that only the carbamate
structure was responsible for the observed enhancing effect.
The reason for the striking difference in the activities of these
compounds remains unclear and is currently under investi-
gation. It could be connected with the following:
1. The unusual structure of the polar head, resulting in a
specific interaction with the stratum corneum components
2. The lability of the carbamate bond in an acidic envi-
ronment and a consequent carbon dioxide release in
the stratum corneum
3. The presence of two hydrophobic chains in the en-
hancer, which is similar to ceramides and could lead to
a better incorporation of the enhancer into a lipid
4. A combination of some or all the three reasons given
To evaluate the permeation-enhancing properties of T12
in greater detail, additional three drugs, including clotrima-
zole, flobufen, and griseofulvin, covering a wide range of
physicochemical properties, were included in the study
(Table I). Substances of a molecular weight up to 353 g/mol
were selected because this weight is similar to that of
oxybutynin, currently the largest drug incorporated in a
patch (20). The polarity of the drugs ranges from log P õ 0
to 5.8; that is, their partition coefficients differ by five orders
of magnitude. The melting points of the drugs vary between
147 and 274-C, and the compounds are of acidic, basic, and
Clotrimazole is an imidazole antifungal of a relatively
high molecular weight and lipophilicity. The addition of 1%
T12 enhanced the permeation of clotrimazole at pH 7.3 and
8.3; however, at pH 9.3, its effect was suppressed. A possible
explanation of such pH-dependent effect is that in aqueous
media, equilibrium between a carbamate, bicarbonate, and
carbonate occurs, and basic conditions would favor bicarbon-
ate and/or carbonate formation (18). This implies that the
alkylcarbamate anion is the most important structural feature
for the enhancing effect of the molecule. However, the be-
havior of T12 under aqueous conditions and the pH depen-
dence of the enhancement effect merit further investigation.
Flobufen is a novel anti-inflammatory drug with immu-
nomodulatory effects (21). It has been included in the study
as an example of a weak acid. Previously, T12 was inactive
when coadministered with an acidic drug, most likely because
of acid-catalyzed decomposition of the carbamate (unpub-
lished observation). However, in a buffered donor vehicle,
T12 enhanced the permeation of acidic flobufen as well as
that of neutral and weakly basic drugs. Similar to clotrima-
zole permeation, a decreased enhancement was observed at
pH 8.7, suggesting an increased bicarbonate/carbamate ratio.
917 Transdermal Permeation Enhancer Transkarbam 12
Griseofulvin is an antifungal substance, produced by the
growth of certain strains of Penicillium griseofulvum. In this
study, it was selected as a representative of large, neutral, and
poorly permeating substances. Using an aqueous receptor
phase, no griseofulvin was detected. The addition of a
relatively high amount of ethanol to the acceptor (40%)
resulted in detectable permeation, although not in all experi-
ments. The flux of griseofulvin without an enhancer was not
further evaluated because the effect of T12 was clearly
shownVan addition of the enhancer resulted in a reproduc-
ible flux of griseofulvin, which is usually concentrated in the
stratum corneum and does not penetrate into lower layers.
The potential of T12 to be enzymatically hydrolyzed in
vitro was shown using porcine esterase. As esterases are
present in the human epidermis (22), the hydrolysis is likely
to occur in vivo as well. Such hydrolysis in the viable
epidermis would prevent the action of the enhancer on the
living cells. The metabolites, e.g., 6-aminohexanoic acid and
dodecanol, are compounds of very low toxicity, which is
another favorable characteristic of this novel enhancer.
Furthermore, the described HPLC method for T12 determi-
nation could be useful in further evaluation of its effect.
Furthermore, toxicity of T12 has been newly evaluated.
Repeated daily dose 28-day dermal toxicity study in rat with
a 14-day treatment-free period revealed no significant clinical
symptoms including site of administration in a group
receiving 0.1% T12, a slight skin erythema at 2% T12, and
a skin erythema and crusts at 10% concentration. After the
end of administration, skin recovered during 5Y14 days.
Significant changes in total leukocyte count were observed,
which correlated with the skin reaction. No changes in body
weight, food consumption, organ weights, red blood cell
parameters, serum chemistry parameters, and no hepato- and
nephrotoxicity have been found in all concentrations used
(23). No toxicity signs were observed in rats in the acute oral
toxicity test in doses of 50, 300, and 2000 mg/kg, neither
during 24 h after administration nor during 14 days of
observation period (24). Index of dermal irritation was 0.25
(category nonirritating) and 1.67 (category slightly irritating)
for 0.5 and 5% suspension of T12 in PG, respectively (25).
T12 did not act as a contact allergen in the closed patch
sensitization test (Buehler’s method) (26). Moreover, T12
had no mutagenic potential in bacteria (the Ames reverse
mutation test) (27) and rats and no cytotoxic effect on the
bone marrow of rats (micronucleus test) (28). All tests were
performed in accordance with the pertinent OECD Guide-
lines for Testing of Chemicals.
In this study, the properties of a promising transdermal
permeation enhancer T12 have been investigated. Comparing
both physicochemical and permeation-enhancing properties
clearly showed that the carbamate salt, not the parent amino
ester DDEAC, was responsible for the high activity of the
compound. The activity of T12 was demonstrated using four
model drugs of different physicochemical properties, and the
susceptibility of the compound to metabolic deactivation into
nontoxic substances was shown in vitro using porcine
We thank Iva Vencovska ´ for the measurement of the IR
spectra, John MacLeod from The Research School of
Chemistry, Australian National University, Canberra, for
measurement of the mass spectra, and Bochemie Ltd.,
Bohumı ´n, Czech Republic, for providing the toxicity data.
This work was supported by the BCentre for New Antivirals
and Antineoplastics^ (1M6138896301) and the Research
Project MSM0021620822 of the Ministry of Education, Youth
and Sport of the Czech Republic.
1. A. C. Williams and B. W. Barry. Penetration enhancers. Adv.
Drug Deliv. Rev. 56:603Y618 (2004).
2. R. B. Stoughton. Enhanced percutaneous penetration with 1-
dodecylazacycloheptan-2-one. Arch. Dermatol. 118:474Y477
3. J. W. Wiechers and R. A. DeZeeuw. Transdermal drug delivery:
efficacy and potential applications of the penetration enhancer
Azone\. Drug Des. Deliv. 6:87Y100 (1990).
4. N. Bu ¨yu ¨ktimkin, S. Bu ¨yu ¨ktimkin, and J. H. Rytting. Chemical
means of transdermal drug permeation enhancement. In T. K.
Ghosh, W. R. Pfister (eds.), Transdermal and Topical Drug
Delivery Systems, Interpharm Press, Buffalo Grove, IL, 1997, pp.
5. B. B. Michniak, M. R. Player, J. M. Chapman, and J. W. Sowell
Sr. In vitro evaluation of a series of Azone analogs as dermal
penetration enhancers. I. Int. J. Pharm. 91:85Y93 (1993).
6. B. B. Michniak, M. R. Player, L. C. Fuhrman, C. A. Christensen,
J. M. Chapman, and J. W. Sowell Sr. In vitro evaluation of a
series of azone analogs as dermal penetration enhancers: III.
Acyclic amides. Int. J. Pharm. 110:231Y239 (1994).
7. B. B. Michniak, M. R. Player, D. A. Godwin, C. A. Phillips, and
J. W. Sowell Sr. In vitro evaluation of a series of Azone analogs
as dermal penetration enhancers: IV. Amines. Int. J. Pharm.
8. B. B. Michniak, M. R. Player, and J. W. Sowell Sr. Synthesis and
in vitro transdermal penetration enhancing activity of lactam N-
acetic acid esters. J. Pharm. Sci. 85:150Y154 (1996).
9. L. C. Fuhrman Jr., B. B. Michniak, C. R. Behl, and A. W.
Malick. Effect of novel penetration enhancers on the transder-
mal delivery of hydrocortisone: an in vitro species comparison.
J. Control. Release 45:199Y206 (1997).
10. K. Va ´vrova ´, J. Zbytovska ´, and A. Hraba ´lek. Amphiphilic
transdermal permeation enhancers; structureYactivity relation-
ships. Curr. Med. Chem. 12:2273Y2291 (2005).
11. P. Doleºal, A. Hraba ´lek, and V. Semecky ´. epsilon-Amino-
caproic acid esters as transdermal penetration enhancing agents.
Pharm. Res. 10:1015Y1019 (1993).
12. A. Hraba ´lek, P. Doleºal, O. Farsa, A. Krebs, A. Kroutil, M.
Roman, and Z. Sˇklubalova ´. w-Amino acid derivatives, processes
of their preparation and their use. U.S. Pat. 6,187,938 (2001).
13. R. Ibuki. Ph.D. Thesis. University of Kansas, Lawrence, 1985.
14. A. Hraba ´lek, P. Doleºal, and K. Pala ´t. Physico-chemical
parameters and skin permeation enhancing effect of w-amino
acid derivatives. In K. R. Brain, K. A. Walters (eds.), Perspec-
tives in Percutaneous Penetration, Vol 7A, STS Publishing,
Cardiff, 2000, p. 70.
15. J. Zbytovska ´, S. Raudenkolb, S. Wartewig, W. Hu ¨bner, W.
Rettig, P. Pissis, A. Hraba ´lek, P. Doleºal, and R. Neubert.
Phase behaviour of transkarbam 12. Chem. Phys. Lipids
16. M. Kuchar, M. Poppova, H. Zunova, E. Knezova, V. Vosatka,
and M. Prihoda. 4-(20,40-Difluorobiphenyl-4-yl)-2-methyl-4-oxo-
butanoic acid and its derivatives. Collect. Czechoslov. Chem.
Commun. 59:2705Y2713 (1994).
17. T. J. Franz. Percutaneous absorption; on the relevance of in vitro
data. J. Invest. Dermatol. 64:190Y195 (1975).
918 Hraba ´lek et al.
18. D. B. Dell’Amico, F. Calderazzo, L. Labella, F. Marchetti, and Download full-text
G. Pampaloni. Converting carbon dioxide into carbamato
derivatives. Chem. Rev. 103:3857Y3898 (2003).
19. K. Va ´vrova ´, A. Hraba ´lek, P. Doleºal, T. Holas, and J.
Klimentova ´. Biodegradable derivatives of tranexamic acid as
transdermal permeation enhancers. J. Control. Release 104
20. M. R. Prausnitz, S. Mitragotri, and R. Langer. Current status
and future potential of transdermal drug delivery. Nat. Rev.,
Drug Discov. 3:115Y124 (2004).
21. V. Panajotova ´, E. Ande ˇrova ´, A. Jandera, and M. Kuchar ˇ.
Pharmacological profile of the novel antirheumatic 4-(20,40-
difluorobiphenyl)-2-methyl-4-oxobutanoic acid. Arzneimittelfor-
schung. 47:648Y652 (1997).
22. W. Montagna. Histology and cytochemistry of human skin IX.
The distribution of non-specific esterases. J. Biophys. Biochem.
Cytol. 1:13Y16 (1955).
23. Report No. 031/04/L. Repeated Dose 28-Day Dermal Toxicity
Study in Rat with a 14-Day Treatment-Free Period. In
accordance with OECD Guidelines for Testing of Chemicals
No. 410. Bochemie Ltd., Bohumı ´n, Czech Republic (2005).
24. Report No. 017/04/L. Acute Oral Toxicity in Rats, Acute Toxic
Class Method. In accordance with OECD Guidelines for Testing
of Chemicals No. 423. Bochemie Ltd., Bohumı ´n, Czech Republic
25. Report No. 025/04/L. Acute Dermal Irritation/Corro-
sionVRabbit. In accordance with OECD Guidelines for Testing
of Chemicals No. 404. Bochemie Ltd., Bohumı ´n, Czech Republic
26. Report No. 024/04/L. Skin Sensitisation in Guinea Pigs. In
accordance with OECD Guidelines for Testing of Chemicals No.
406. Bochemie Ltd., Bohumı ´n, Czech Republic (2004).
27. Report No. 027/04/L. Bacterial Reverse Mutation Test. In
accordance with OECD Guidelines for Testing of Chemicals
No. 471. Bochemie Ltd., Bohumı ´n, Czech Republic (2004).
28. Report No. 023/04/L. Micronucleus Test in Rats. In accordance
with OECD Guidelines for Testing of Chemicals No. 474.
Bochemie Ltd., Bohumı ´n, Czech Republic (2004).
919 Transdermal Permeation Enhancer Transkarbam 12