Eudragit E100 as a drug carrier: The remarkable affinity of phosphate ester for dimethylamine

Article abstract of DOI:10.1021/mp300282f

Therapeutic agents containing phosphate groups in their molecules have increasing therapeutic impact. The object of this study was to characterize the cationic polyelectrolyte Eudragit E100 (EuE100) as a carrier for drugs containing phosphate groups, using dexamethasone phosphate (DP) as a model. A series of EuE100-DP complexes was obtained by acid-base reaction in which DP neutralized 12.5-75% of the basic groups of EuE100. The solids obtained after solvent evaporation revealed by spectroscopic characterization the complete reaction between the components through the ionic interaction between the amine groups of EuE100 and the phosphate groups of DP. The reversibility of the counterion condensation, evaluated through the proton-withdrawing effect produced by the ionic exchange generated by titration with NaCl, showed a remarkable high affinity between EuE100 and DP. In line, drug delivery in bicompartimental Franz cells toward water as receptor medium was very slow (2% in 6 h). However, it was increased as water was replaced by NaCl solution, which upon diffusion generates ionic exchange. A sustained release of DP with noticeable zero order kinetics accounted for a remarkable high affinity, mainly due to the electrostatic attraction. The release rate remains constant regardless of the saline concentration of the media. Besides, the delivery control is maintained even in gastric simulated fluid, a property not informed previously for EuE100 complexes.

Full text of DOI:10.1021/mp300282f

Article
pubs.acs.org/molecularpharmaceutics
Eudragit E100 as a Drug Carrier: The Remarkable Affinity of
Phosphate Ester for Dimethylamine
M. L. Guzman, R. H. Manzo, and M. E. Olivera*
́
Department of Pharmacy, Faculty of Chemical Sciences, Pharmaceutical Technology Research Unit (UNITEFA-CONICET),
National University of Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria (5000), Cordoba, Argentina
́
́
*
S Supporting Information
ABSTRACT: Therapeutic agents containing phosphate groups in their molecules
have increasing therapeutic impact. The object of this study was to characterize the
cationic polyelectrolyte Eudragit E100 (EuE100) as a carrier for drugs containing
phosphate groups, using dexamethasone phosphate (DP) as a model. A series of
EuE100-DP complexes was obtained by acid−base reaction in which DP neutralized
1
2.5−75% of the basic groups of EuE100. The solids obtained after solvent
evaporation revealed by spectroscopic characterization the complete reaction between
the components through the ionic interaction between the amine groups of EuE100
and the phosphate groups of DP. The reversibility of the counterion condensation,
evaluated through the proton-withdrawing effect produced by the ionic exchange
generated by titration with NaCl, showed a remarkable high affinity between EuE100
and DP. In line, drug delivery in bicompartimental Franz cells toward water as receptor
medium was very slow (2% in 6 h). However, it was increased as water was replaced by NaCl solution, which upon diffusion
generates ionic exchange. A sustained release of DP with noticeable zero order kinetics accounted for a remarkable high affinity,
mainly due to the electrostatic attraction. The release rate remains constant regardless of the saline concentration of the media.
Besides, the delivery control is maintained even in gastric simulated fluid, a property not informed previously for EuE100
complexes.
KEYWORDS: phosphate ester, controlled release, cationic polymethacrylate
1
2
INTRODUCTION
compact nucleic acid nanoparticles. In some cases, it has also
been reported that cationic polymers can help nucleic acid
therapeutics survive the different extracellular and intracellular
■
The use of polymers as carriers in drug delivery has rapidly
developed over the last decade. The design of dosage forms for
new or existing drugs poses challenges in terms of formulation
needs and the desired release profiles.
Polyelectrolytes (PEs) under the form of ionic exchange
resins (insoluble PE) or dispersible hydrophilic polymers
1
14,15
pathways during cellular uptake and intracellular trafficking.
In all of these cases, little (if so) explanation about the
polymer−drug interactions was given. In most of them, the
negative charges interacting with the basic PEs are provided by
phosphate moieties. Phosphate groups are also present in a
variety of drugs and drug derivatives. Therefore, the study of
the interactions that occur between the basic groups of PE and
phosphate acidic groups is relevant.
Eudragit E100 (EuE100) is a cationic polymer based on
dimethylaminoethyl methacrylate (DMAEMA) and other
neutral methacrylic acid esters. EuE100 is widely used in oral
and topical formulations and is generally regarded as nontoxic,
nonirritant, and essentially safe in humans. It is currently used
as coating in solid pharmaceutical dosage forms for taste
masking and for protection. Its use as a dissolution modifier
and as a granulating agent has also been explored.
transdermal drug delivery systems were also reported.
(
soluble PE) have been largely used in pharmaceutical
2
,3
formulations.
The unique properties arising from the
interaction of PE with inorganic or organic counterions have
been exploited for a variety of purposes such as drug delivery
modulation, taste masking, and drug compatibility/stability
4
−6
improvement, among others.
The knowledge about the factors that determine the
interaction between ionic or ionizable drugs and PE is relevant
in the design of pharmaceutical dosage forms.
Basic PEs, such as chitosan, chitosan derivatives, poly-
glycoamideamine)s, and cationic polymethacrylates, are able
to interact with oppositely charged compounds, which results
in spontaneous association due to the formation of strong but
reversible electrostatic links.
16
(
1
7,18
Several
1
9
7
−12
Modified drug release is achieved due to the interaction
The interaction between the
basic groups of the PEs (mostly amines) with carboxylic acid
1
3
moieties was reported. Besides, several potentialities are
claimed for basic PEs concerning their ability to interact with
peptides or to act as nonviral delivery vehicles for plasmid
Received: August 17, 2011
Revised: July 16, 2012
Accepted: July 18, 2012
Published: July 18, 2012
9
−12
DNA.
They have also showed a capacity to bind and to
©
2012 American Chemical Society
2424
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
between reactive groups of copolymer pairs in combination
with polyanionic polymers containing carboxylic acids such as
EuL100 or EuS100.
Etilfarma S.A. (Buenos Aires, Argentina). DP was obtained by
neutralization of the sodium salt (DPNa ) with 1 M HCl
2
20
solution. The obtained solid melts at 205 °C. Benzoic acid (BA,
Parafarm ) and diclofenac (DI, Cicarelli ) pharmaceutical grade
were used. Simulated gastric fluid without pepsin (SGF) was
We have previously reported some results in relation to the
properties of the complexes between the cationic polymer
13
23
EuE100 and some drugs possessing carboxylic acid groups.
prepared according to USP 34 Ed.
−
When Cl is used as an additional counterion, clear optically
isotropic aqueous dispersions are produced. This increase in
aqueous compatibility allows the loading of acid drugs and
generates systems with a high condensation ratio between the
drugs and the polymer. This property allows the increase of the
aqueous compatibility of low solubility acid drugs in the range
of pH in which solubility is determined by the undissociated
species. They also have the capability to control the release rate
since EuE100 behaves as a carrier that slowly releases the
studied model drugs containing carboxylic acid groups as it is
dispersed in water but increases the delivery rate in saline
solutions simulating biological fluids. All of these properties
make these complexes interesting for developing systems for
nonparenteral routes in which a controlled release is desired. At
present, studies concerning the interaction of EuE100 with
moieties containing phosphate ester groups are not reported.
This work is focused on the ionic interaction between the
amine groups of EuE100 and the phosphate groups of a
selected model drug, dexamethasone phosphate (DP), whose
structures are presented in Figure 1. This study is relevant not
Preparation of Solid Complexes. Before complexation,
EuE100 was milled and passed through 40 and 70 mesh sieves.
The equivalents of amine groups per gram of EuE100 (3.10 ×
−3
10 ) were assayed by acid−base titration, which is equivalent
to 23.8% of the ionogenic group DMAEMA in the polymer
24
(theoretical value 20.8−25.5).
A series of complexes were prepared by dispersing EuE100 in
a mortar with 8 mL of ethanol per gram of polymer. Variable
amounts of HCl 0.1 M were added to obtain an initial partial
neutralization of the amine groups of EuE100. The mixture was
kept for 30 min at room temperature to favor hydration and
relaxation of the polymer. After that, DP was added as a powder
to neutralize a given fraction of the amine groups of EuE100.
Then, the solvent was evaporated under vacuum at 45 °C. This
series was regarded as EuE100-Cl DP , in which (x) and (y)
(
x)
(y)
refer respectively to the mole percent of the incorporated HCl
or DP. Additionally, a complex EuE100-DP(25), in which HCl
was not added, was prepared. Physical mixtures (PM) were
prepared by mixing in a mortar EuE100 and DP in the same
proportion used to obtain the complex.
Spectroscopic and Thermal Characterization. Solid
products EuE100-DP(25), EuE100-Cl(25−75)DP(12.5−75), EuE100,
DP, and their respective PM, were characterized through
Fourier transform infrared spectroscopy (FTIR), thermal
analysis, and X-ray powder diffraction (XRPD).
FTIR. The infrared spectra of the samples dispersed at 1% of
DP in KBr discs were recorded in a NICOLET FTIR (360
FTIR ESP, Thermo Nicolet, Avatar) spectrometer. The
−1
samples were scanned from 4000 to 400 cm . The recording
conditions were resolution, 8.0; sample scan, 40. Data were
analyzed using OMNIC software.
Thermal Analysis. The samples were subjected to differ-
ential scanning calorimetry (DSC) and thermogravimetric
analysis (TGA), from room temperature to 350 °C. The
DSC standard cell of an A2920 MDSC (TA Instruments)
equipped with a data station (Universal Analysis, TA
Instruments) was used to determine DSC curves. The
temperature axis and the cell constant of the DSC cell were
calibrated with indium (24 mg, 99.99% pure, peak maximum at
1
56.66 °C, and heat of fusion 28.71 J/g, cell constant 1.2375).
The samples (0.9−1.7 mg) were heated in nonhermetic
aluminum pans with a pine hole, under nitrogen flux (60
mL/min). Samples were run at 10 °C/min ramps. The areas of
the endothermic peaks were found by drawing a line from the
point where the thermogram first departs from the baseline
Figure 1. Structural formula of EuE100 monomer (up) and DP
(
down).
(
onset) to the point where the baseline was resumed following
only to increase the potentiality of EuE100 to load new groups
of drugs but also to approach the study of the interaction with
phosphate groups in DNA molecules, nucleotides, or plasmids
which have an increasing therapeutic impact. In this context,
EuE100 has been used in techniques for precipitation/isolation
fusion (recovery). The fusion and dehydration temperatures
were taken as the extrapolated onset temperature of the
endothermic peak. A 2950 TGA HR thermogravimetric
analyzer (TA Instruments) linked to a data station was used.
The samples (approximately 2 mg) were placed in open
aluminum pans and heated under the same conditions used in
their respective DSC analysis.
For the determination of glass transition temperature (T
additional DSC experiments were carried out. Samples (4−7
mg) of EuE100, EuE100-Cl , and EuE100-Cl DP
21,22
of proteins, protein encapsulation, among others.
MATERIALS AND METHODS
■
g
)
Materials. Poly(butyl methacrylate, (2-dimethyl
aminoethyl)methacrylate, methyl methacrylate) 1:2:1 (Eudragit
E100, Pharmaceutical grade, Rohm, Germany) was a gift from
(
25−100)
(25)
(25−75)
were accurately weighed into aluminum pans, which were
2
425
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
sealed and perforated. The samples were heated (20 °C/min),
under nitrogen flux (60 mL/min) from room temperature to
0.23, and 1.20 ± 0.05 (in water, NaCl, and SGF, respectively)
at the end.
1
00 °C and maintained at this temperature for 15 min to avoid
To confirm sink conditions, solubility determinations of DP
were conducted. An excess of DP was placed into suitable
stoppered containers and added with SGF, water, or 0.9%
NaCl. The samples (in triplicate) were immersed in a
thermostatized water bath at 37 °C and periodically shaken.
Once the equilibrium was reached, the pH of the supernatant
was recorded. Aliquots of the filtrate, properly diluted with the
same solvent, were analyzed by UV spectrophotometry at the
maximum wavelengths (λ = 241 nm). The solubility of DP was
moisture effect on the T measurement. Then the samples were
cooled to 0 °C and reheated (20 °C/min) to 200 °C. The Tg
was determined from the second heating cycle.
g
XRPD. This technique was used to identify crystalline phases
and to qualitatively examine changes in crystallinity. Measure-
ments were done with a Rigaku Miniflex diffractometer (The
Woodlands, TX) having a copper target X-ray tube, equipped
with specific software (Standard monitoring 3.2), operated in
scan mode over 5−80° 2θ, steps of 0.05, and scan speed of
3
=
0
.01 ± 0.03 (water, pH = 1.30 ± 0.05), 3.04 ± 0.01 (NaCl, pH
1.13 ± 0.02), and 1.22 ± 0.09 mg/mL (SGF, pH = 0.97 ±
.04). The BA solubility at 25 °C in 0.15 M KCl has been
0.66° 2θ/s. The accelerating voltage was 30 kV, and the current
was 15 mA.
1
1
H NMR Experiments. Solution H NMR spectra were
recorded at 25 °C on Bruker Advance II Ultra Shield TM
spectrometer operating at 400.16 MHz. A sample of EuE100-
Cl(30)DP(40) that had a final DP concentration of 9 mM was
prepared by dispersing the complex in a mixture of H O/D O
26
reported as 3.2 mg/mL (pH = 1) and 29.5 mg/mL (pH = 5).
Hence, under the conditions used (volume of the receptor
compartment, sampling frequency, and concentration of drugs
in the donor compartment), sink conditions (defined as the
volume of medium at least greater than three times that
2
2
80:20. In addition, samples of both DP and EuE100-Cl(30) at
27
required to form a saturated solution of a drug substance )
the same respective concentration as that of the complex were
recorded. The ionic strength was regulated by adding NaCl to
DP and EuE100-Cl(30) samples to obtain values equivalent to
that of the EuE100-Cl(30)DP(40) dispersion. TSP (trimethylsi-
lylpropionic acid-d4, sodium salt) was used as an internal
reference. Assignments of DP were done by comparing the
prevailed in all studies.
In addition, a set of experiments were run in which NaCl was
added to the donor compartment and the receptor was filled
with water. In these cases, the dispersions of EuE100-
Cl(25)DP(38) or EuE100-Cl(25)BA(50) were added with 0.5 mL
of a NaCl solution containing between 50 and 750% of the
equivalents of DP or BA present in the dispersions. All of the
assays were run in triplicate and followed for 6 h.
25
values with those in the literature.
DP Displacement by Ion Exchange. The effect of the
addition of sodium chloride on ionic equilibrium of complex
dispersions was evaluated through the titration of 20 mL of an
EuE100-Cl(25)DP(40) aqueous dispersion (3.5 × 10− mole of
DP) with NaCl 0.9%. The pH of the dispersions was plotted
against NaCl concentration, referred to as percentage of DP in
the complexes. Dispersions of complexes of EuE100-
Cl(25)BA(40) and EuE100-Cl(25)DI(40), at the same molar
concentration as that of DP, were titrated and used for
comparison as a model systems containing drugs with
carboxylic acid groups.
5
RESULTS AND DISCUSSION
■
Table 1 reports the series of EuE100-Cl DP complexes
(
x)
(y)
prepared in the solid state, in which the proportions of DP and
HCl were systematically modified. The complexes were easily
obtained in the solid state as described in the experimental
Table 1. EuE100-Cl DP Complexes Obtained in Solid
(
x)
(y)
State
Drug Release. Aqueous dispersions of complexes of
EuE100-Cl(25)DP(38) and EuE100-Cl(25)DP(50) (4 mL, equiv-
alent to 22 and 28 mg of DP, respectively) were subjected to
drug release analysis. The release rate from an aqueous solution
total
neutralization
(%)
a
a
EuE100-
Cl(x)DP(y)
HCl
DP
pH of 1% (w/v)
aqueous dispersion
a
,
b
(%)
(%)
EuE100-
75
25
100
100
100
75
1.96
3.17
c
with an equivalent concentration of DPNa was used as a
2
Cl(75)DP(25)
reference. A dispersion of EuE100-Cl(25)BA(50), at the same
molar concentration as that of DP (4 mL, equivalent to 7.87 mg
of BA), was used for comparison.
EuE100-
Cl(50)DP(50)
50
25
50
25
30
25
25
25
50
75
25
50
40
38
25
EuE100-
Cl(25)DP
EuE100-
Cl(50)DP
EuE100-
(75)
(25)
Experiments were performed in a modified Franz diffusion
assembly at 37 ± 1 °C. A semipermeable acetate cellulose
membrane (Sigma 12 000) was placed between the donor and
the receptor compartments. Complex dispersions were placed
in the donor compartment. The receptor compartment was
filled with 70 mL of water, 0.9% NaCl solution, or SGF and
stirred with a Teflon-coated magnetic stirring bar. At selected
times, 4 mL aliquots were withdrawn and replaced by the same
volume of receptor medium, prewarmed at 37 °C. Data were
corrected for dilution. DP and BA concentrations were
determined by UV spectroscopy (Thermo-Electronic Corpo-
ration, Evolution 300 BB, England) at 241 nm and 230 nm,
respectively.
4.45
4.88
5.12
5.45
5.90
6.08
c
75
Cl(25)DP(50)
EuE100-
70
Cl(30)DP
EuE100-
Cl(25)DP
EuE100-
(40)
(38)
63
50
Cl(25)DP(25)
EuE100-
Cl(25)DP
12.5
25
37.5
25
(12.5)
EuE100-
DP(25)
a
The pH values of the receptor compartments were 6.15, 5.80,
and 1.20 (in water, NaCl, and SGF, respectively) at the
beginning of the release experiments and 4.98 ± 0.31, 5.80 ±
Expressed as equivalents of dimethylamine groups of EuE100.
Considering 1 equiv/mol. pH values not registered due to the low
b
c
solubility of the 1% (w/v) complexes dispersions.
2
426
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
section. Most of the systems are hydrophilic enough to obtain
1
% (w/v) dispersions.
Spectroscopic and Thermal Characterization of the
Complexes. EuE100 is a strong polybase and acts as proton
28
acceptor. The average pK of the basic monomer is 8.4.
a
Besides, DP presents values of pK and pK of 1.89 and 6.4,
a1
a2
2
9
respectively.
In the FTIR analysis, the indicative bands of the acid−base
interaction were investigated for changes arising as a result of
the association between EuE100 and DP. The appearance of
new bands was also investigated. The regions of the stretching
modes associated with the phosphate group of DP (P−OH st,
−
and asymmetric st mode of the POO ) and the bands of
dimethylamine groups of EuE100 were analyzed in the
complexes, DP, EuE100, and their PM. The FTIR spectra of
the solid complexes showed some remarkable differences when
they were compared to that of the reactants (Figure 2).
The absorption bands of the nonprotonated dimethylamine
−1
groups appear at 2770 and 2816 cm in EuE100 and the PM.
In the complexes, these bands are significantly reduced which
indicates the presence of protonated and nonprotonated
3
0,31
dimethylamine groups.
The reduction is related to the
neutralization percentages of the samples, and the bands
disappear when all of the dimethylamine groups of EuE100 are
protonated in EuE100-Cl(50)DP(50). Interestingly, the dimethyl-
amine bands of the complex EuE100-Cl(50)DP(25) have a
stronger intensity than those of the complex EuE100-
Cl(25)DP(50), although they have the same neutralization
percentage. This suggests that, in the solid state, each
phosphate group could be interacting with more than one
dimethylamine group of EuE100. This is possible since the pKa2
of this group is about 2 units lower than that of EuE100.
−1
In DP, a band at 970 cm ascribed to the P−OH st of the
32
phosphate group was observed. This band is also present in
−1
the PM (973 cm ) but disappears in all of the complexes and
−
1
DPNa . Besides, the appearance of a new band at 987 cm
2
−
associated with the asymmetric stretching mode of POO
groups was observed in all of the complexes and DPNa2
Figure 2). This points out the presence of phosphates
(
ionically bound to protonated amines. The absence of
unreacted DP in the complexes also suggests that the
preparation method allows a complete reaction between
EuE100 and DP and all of the dimethylamine groups of
EuE100 would be able to interact with the phosphate groups,
giving complexes with high loading capacity.
Representative XRPD patterns are shown in Figure 3. The
patterns clearly show the crystalline character of DP given by
different peaks at fixed angles. Their specific values are
presented in the Supporting Information. On the other hand,
EuE100 shows an amorphous character without defined peaks
in the XRPD pattern. The PM showed the characteristic peaks
of DP and a profile similar to its sum with EuE100. In line with
the ionic interaction observed by FTIR, no reflections of
crystalline DP were present in the complexes, which showed
characteristic profiles of amorphous compounds. The dis-
appearance of the peaks of DP suggests that it exists in an
amorphous form and a complex was formed between EuE100
and DP. These results are in line with those observed by FTIR
Figure 2. Representative FTIR spectra of DP, DPNa , EuE100, PM,
2
and some EuE100-Cl DP complexes. Bands corresponding to: ∗,
(x)
(y)
stretching of C−H groups from the dimethylamine N−CH of
3
−1
EuE100 (2770 and 2816 cm ); ▲, symmetric and assymmetric
−1
stretching of P−OH group (970 cm ); ●, symmetric stretching of
−
−1
POO group (987 cm ). For clarity, only some representative spectra
were included in the figure.
be related to some crystalline points in the samples. However,
13
they are attributable to neither the DP nor EuE100.
Figure 4 shows the DSC and TGA thermograms of EuE100,
DP, their PM, and a representative complex. DP presents an
endothermic peak (onset: 57.17 °C, maximum peak: 94.04 °C,
recovery: 118.42 °C) paralleled by 0.49% of mass loss in TGA
which was assigned to water loss. The melting endothermic
peak (onset: 185.67 °C, maximum peak: 204.94 °C) was
followed by an exothermic event above 211 °C, attributable to a
3
,13
and those previously reported.
Notice that three sharp
reflections at 37°, 45°, and 70° 2θ appear in the complexes in a
region where crystalline DP has no signals. This behavior was
previously observed in some EuE100-DI complexes and should
2
427
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
decomposition process. In the complexes, the melting peak of
DP was absent confirming the complete reaction between the
EuE100 and the DP in all of the proportions evaluated. All of
the complexes exhibited decomposition after 240 °C. The PM
shows a small endothermic peak corresponding to DP melting,
which is substantially lower than the expected one for the
amount of DP in the sample. This behavior could be related to
partial reaction, probably favored by the assay heat. In fact,
from the ratio between the DP experimental melting enthalpy
in the PM and the expected one for the pure form, the
proportion of material that is in molecular dispersion or in
crystalline form could be estimated (92% and 8%, respectively).
Measuring the T of solid dispersions can give a wealth of
g
information pertaining to the physical properties of these
systems, such as the physical state (amorphous or not) of the
drug/carrier, miscibility of the drug with the carrier, or potential
3
3,34
for specific interactions between them.
In agreement with
published results, EuE100 showed a T of 44.5 °C and
g
35
decomposition after 320 °C. The drug-loaded systems,
Figure 3. X-ray powder diffraction patterns of some EuE100-
EuE100-Cl(25)DP(25−75), showed one single T indicating that
g
Cl DP complexes, the reactants, and their PM.
DP is molecularly distributed. As DP percentage goes from 25
(x)
(y)
to 75%, a significant increase in T was observed (Figure 5)
g
Figure 5. Glass transition temperatures (T ) of EuE100 (○), EuE100-
g
Cl(25−100) (⧫) and EuE100-Cl(25)-DP(25−75) (●). The x-axis shows the
total DMAEMA groups of EuE100 neutralized by Cl or DP.
suggesting ionic interactions between phosphates and proto-
nated amines as observed by FTIR. A similar behavior was
informed after salt formation between poly(acrylic acid) and
three basic model compounds, which resulted in high T
g
36
values. The heteromolecular forces in the salt are ionic
while homomolecular interactions occurring in the pure
polymer are not. Since ionic interactions are stronger, this
leads to an increase in density and hence a decrease in free
volume. Higher temperatures, therefore, are needed to bring
such material above its T . The neutralization of EuE100 with
g
HCl to obtain EuE100-Cl(25−100) showed the same tendency
(
Figure 5). Interestingly, T values of EuE100-Cl(25)DP(25−75)
g
were higher than EuE100-Cl(25−100) at the same complexation
ratio. It has been shown that the size of the electrostatic forces
37
in amorphous salts determines the rise of the T values,
g
suggesting that the magnitude of the electrostatic forces
between the dimethylamine and phosphate groups are stronger
−
than dimethylamine and Cl .
1
1
H NMR Experiments. As expected, when H NMR of
Figure 4. DSC (black) and TGA (red) curves for: (a) DP, (b)
EuE100, (c) EuE100-Cl(25)DP(25); (d) EuE100-DP(25) PM. The arrow
shows the melting of DP in the PM.
EuE100, DP, and EuE100-Cl(30)DP(40) spectra were compared
(
Figure 6), EuE100 signals were wide and unresolved due to
38
efficient spin-net relaxation, usually observed in this systems.
1
On the contrary, H NMR spectrum of DP is well-resolved.
2
428
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
1
Figure 6. H NMR spectra of DP, EuE100-Cl(30), and EuE100-Cl(30)-DP(40) in H O/D O (80:20).
2
2
The signals ascribed to the vinyl conjugated system between 6.2
and 7.4 ppm and the three methyl signals with different
environments in C-18, C-19, and C-22, which are close to data
Figure 7 shows the increase of pH against the percentage of
NaCl relative to the moles of DP in the complexes. In this way,
25
1
found in literature, were observed. In the H NMR spectrum
of the complex, important differences can be observed in DP
and EuE100 signals, the widening of the vinyl system signals of
DP being the most noticeable one. It suggests a reduction in its
relaxation times due to the interaction with the polymeric net.
Important modifications are also observed between 1 and 4
ppm in shape and location of the signals corresponding to
EuE100, which indicate a perturbation in the environment
owing to the presence of DP. All of the observations are strong
evidence of the interaction/association between EuE100 and
DP.
DP Displacement by Ion-Exchange. The titration with
NaCl of the fraction of drug condensed with the PE has been
successfully performed in dispersions of complexes of acidic PE
with basic drugs as well as in dispersions of complexes of
Figure 7. pH changes in aqueous dispersions of the complexes due to
displacement of drugs by ion exchange with NaCl.
6
,13
EuE100 and some drugs possessing carboxylic acid groups.
The addition of NaCl to EuE100-Cl DP systems should
(x)
(y)
produce the equilibria depicted in eq 1. Then, under conditions
in which the shift to the right of equilibrium shown in eq 2
would be significant to produce a proton withdrawing effect
the overall proton withdrawing effect produced by the addition
of NaCl on the complexes EuE100-Cl(25)DP(40) should be
ascribed not only to the shifting of equilibrium in eq 2 but also
to a secondary contribution due to the shifting to the right of
equilibria in eq 3. Previous studies showed that pH changes
produced by ion-exchange of EuE100 and NaCl are
+
that overcome the deprotonation of R-N(CH ) H , a plot of
3
2
pH versus [NaCl] would account for the degree of DP
displacement.
13
negligible. Therefore, it is clear that the ionic exchange
+
−
−
[
EuE100H D−O−PO H ] + NaCl
between Cl and anionic species of DP produces the main
3
contribution to the pH shift. For comparison, Figure 7 also
shows the profiles obtained from complexes of EuE100-
Cl(25)BA(40) and EuE100-Cl(25)DI(40) as systems containing
model drugs with carboxylic acid groups. It has been shown
that these complexes have a degree of counterion condensation
+−
−+
⇌
[EuE100H Cl] + D−O−PO H Na
(1)
3
D−O−PO3 − ⇌ D−O−PO H + OH
2
−
−
3
13
−
as high as 97.9%, which was reversible by titration with NaCl.
⇌
D−O−PO H + OH
(2)
(3)
3
2
Notice that, in the EuE100-Cl(25)DP(40) complex, a plateau is
reached after addition of more than 800% of NaCl while, in
complexes with carboxylic acid moieties, it is achieved after only
EuE100H ⇌ EuE100 + H+
+
2
429
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
00% NaCl. This fact would suggest that the interaction
between EuE100 and DP is stronger than that with model
drugs with carboxylic acid groups.
Article
2
are able to diffuse (Figure 9b). The release in SGF drives to an
additional increase in DP release rate due to a shift to the right
in eq 2. However, the release is still controlled (Figure 8a). In
addition, the delivery rate from EuE100-Cl(25)DP(38) and
EuE100-Cl(25)DP(50) approaches zero order kinetics, and no
burst effect was observed.
Notice that, the increase of DP release rates in the complexes
due to the replacement of water by NaCl solution in the
receptor compartment was markedly lower than those observed
with BA complexes (Figure 8b). It was also lower than those
observed in aqueous dispersions of EuE100 complexes with
Drug Release. Preliminary experiments (unshown results)
revealed that tableted EuE100-Cl DP solid complexes
(
x)
(y)
rapidly dissolve in acidic media as well as in water when
assayed by dissolution apparatus. As a consequence, the
released DP cannot be easily differentiated from complexed
DP. Based on that, release toward biorelevant fluids in
bicompartimental Franz cells from complex aqueous dis-
persions (rather than in the solid state) was selected to study
the release behavior of the complexes. The same approach has
been previously used by other authors to obtain mechanistic
and quantitative information regarding rate and kinetics of
13
other drugs containing a carboxylic acid groups and with the
highly condensed acid PE-basic drug aqueous dispersions of
40
carbomer. In contrast, EuE100-Cl(25)BA(50) dispersions failed
to control release of BA in acidic media showing the same
release profile as its reference solution. This behavior is in line
with that observed in the displacement by ion-exchange,
suggesting that the interaction of phosphate groups with
EuE100 is stronger than those of the carboxylic acid groups. In
agreement with FTIR observations, this could be due to the
ability of phosphate groups in DP to interact with more than
one protonated dimethylamine group from EuE100, whereas in
the case of drugs with carboxylic acid groups such as BA, each
molecule would establish an ionic interaction with only one
protonated dimethylamine group. Therefore, the drug release
rate would be much slower in the case of E100-Cl DP
(y)
3
9−41
release.
Diffusion of DP from the complexes EuE100-Cl(25)DP(38)
and EuE100-Cl(25)DP(50) was evaluated. Dispersions of complex
EuE100-Cl(25)BA(50), as a model system containing a drug with
a carboxylic acid group, were also assayed for comparison. It
was selected because BA has good water solubility, and hence
sink conditions are warranted. When the receptor compartment
was filled with water, a fast diffusion of DP from the reference
solution was observed (Figure 8). However, release rates of DP
(x)
complexes. To get further information to clarify this point, a set
of comparative experiments between the release behavior of
EuE100-Cl(25)DP(38) and EuE100-Cl(25)BA(50) dispersions
added with increasing proportions of NaCl in the donor
compartment was performed. The results shown in Figure 10
clearly demonstrate the remarkable affinity of EuE100-
Cl(25)DP(38) whose release rate remains constant regardless of
the saline concentration.
It should be considered that a PE-drug complex dispersed in
water creates a microenvironment with a number of properties
such as electrokinetic potential, intra- and interchain inter-
actions, and a number of interactions with the bulk medium
that are expected to be modified during the process of delivery.
In fact, polybasic polymers as chitosan and EuE100 exhibit a
pH-sensitive behavior due to the large quantities of amine
groups on their chains. Besides, their solubility in aqueous
media also depends on temperature and ionic strength of the
42,43
dissolving medium.
Therefore, the interaction of the
complexes with different physiological fluids (due, for example,
to different administration routes) can have a significant impact
on its performance, and further detailed studies will be
necessary to get a more complete description of the delivery
in selected conditions. In addition, for drugs having phosphate
ester moieties, the enzymatic action of phosphatases is an
additional factor which could influence the in vivo release rate.
Figure 8. Delivery of (a) DP and (b) BA from EuE100-Cl(25)-DP(50)
and EuE100-Cl -BA
complexes toward: (▲) water, (■) SGF,
(
25)
(50)
and (⧫) NaCl 0.9%; (●) DP and BA reference solutions toward water.
from the aqueous dispersions of both complexes were
substantially slower. The diffusion of anionic species of DP
from the complexes is substantially prevented by the macroion
electrostatic attraction; then, the release should occur mainly
through the diffusion of the free DP species (see Figure 9a).
Therefore, the slow release of DP observed in Figure 8a (<2%
in 6 h) is a consequence of the low concentration of free DP
due to the counterionic condensation on one hand and the high
degree of ionization of the phosphate group of DP on the
other. As also depicted in Figure 8, when water in the receptor
compartment was replaced by a solution of NaCl, release rates
from the DP complexes were increased as a consequence of the
CONCLUSIONS
■
The methodology employed to prepare the complexes allows
the complete reaction of the components. The products
obtained are amorphous solids with a high loading capacity. In
the complexes EuE100-Cl DP a remarkable high affinity
(
x)
(y)
between phosphate ester groups and dimethylamine groups was
evident, which is also higher than that observed for compounds
interacting through carboxylic acid groups. The interaction
between EuE100 and the phosphate groups of DP is ionic and
probably involves the two acidic groups of the molecule. Such
results also support the conclusion that the main contribution
+
−
diffusion of Na and Cl to the donor compartment generating
a ionic exchange that produces free DP and also its salts, which
2
430
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
Figure 9. Ionization equilibria of dimethylamine groups of EuE100 (black) and phosphate groups of DP (blue) in aqueous dispersions of complexes
EuE100-Cl DP : (a) in water, (b) produced by the addition of NaCl. EuE100 and DP are subjected to the acid−base equilibria described in eqs 1
(x)
(y)
and 2. The hydrolysis of DP released produces an increase in pH. The dashed line represents the semipermeable membrane which can be passed
through by the free DP species. The bigger the arrow, the higher the diffusion rate.
affinity between the dimethylamine groups of EuE100 and
phosphate groups could be exploited to develop new systems
with therapeutic agents containing phosphate groups in their
molecules, such as DNA, nucleotides, or plasmids.
ASSOCIATED CONTENT
■
*
S
Supporting Information
Specific values for intensity of X-ray powder diffraction for DP.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
Figure 10. Release of DP (filled symbols) and BA (empty symbols)
from EuE100-Cl DP and EuE100-Cl BA aqueous disper-
́
*National University of Cordoba, Pharmaceutical Technology
Research Unit (UNITEFA-CONICET), Department of
Pharmacy, Faculty of Chemical Sciences, Haya de la Torre y
(
25)
(38)
(25)
(50)
sions added with (□) 75%, (○) 100%, (△) 150%, (◊) 200%, and (●)
50% of NaCl in the donor compartment. NaCl is referred to as the
7
́
Medina Allende, Ciudad Universitaria (5000), Cordoba,
Argentina. Fax: +54-351-4334127. Phone: +54-351-4334163
ext 111. E-mail: meoliver@fcq.unc.edu.ar.
molar percentage of DP or BA in the complexes.
Notes
to the overall interaction arises from the electrostatic attraction.
Aqueous dispersions of EuE100-Cl DP are physically stable
The authors declare no competing financial interest.
(x)
(y)
systems with a high degree of counterion condensation
between EuE100 and DP. EuE100 behaves as a carrier loaded
with DP and could function like “smart” systems designed to
release slowly the drug as it is dispersed in water. The delivery
rate increases in saline solutions simulating biological fluids. A
noticeable zero order kinetics with no burst effect was also
observed. More research is necessary to determine if the high
REFERENCES
■
(
1) Fu, Y.; Kao, W. J. Drug release kinetics and transport mechanism
of non-degradable and degradable polymeric delivery systems. Expert
Opin. Drug Delivery 2010, 7, 429−433.
(2) Jantzen, G. M.; Robinson, J. R. Ion-exchange systems. Modern
Pharmaceutics 1996, 3, 592−593.
2
431
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
(
3) Jimenez-Kairuz, A. F.; Llabot, J. M.; Allemandi, D. A.; Manzo, R.
(23) United States Pharmacopoeia. The National Formulary and
Dispensing Information (USP 34-NF 29); USP: Rockville, MD, 2011.
(24) Specifications and test methods for EUDRAGIT E 100,
H. Swellable drug-polyelectrolyte matrices (SDPM) Characterization
and delivery properties. Int. J. Pharmaceutics 2005, 288, 87−99.
(
4) Ramírez Rigo, M. V.; Allemandi, D. A.; Manzo, R. H. Swellable
̈
EUDRAGIT E PO and EUDRAGIT E 12,5. Degussa. Rohm GmbH &
drug−polyelectrolyte matrices (SDPM) of alginic acid: Character-
Co. KG, 2011. http://eudragit.evonik.com/product/eudragit/
Documents/evonik-specification-eudragit-e100,-eudragit-e-po-and-
eudragit-e-12,5.pdf.
ization and delivery properties. Int. J. Pharmaceutics 2006, 322, 36−43.
(
5) Agresti, C.; Tu, Z.; Ng, C.; Yang, Y.; Liang, J. F. Specific
interactions between diphenhydramine and α-helical poly(glutamic
acid)A new ion-pairing complex for taste masking and pH-
controlled diphenhydramine release. Eur. J. Pharm. Biopharm. 2008,
(25) Midelfart, A.; Dybdahl, A.; Muller, N.; Sitter, B.; Gribbestad, I.
S.; Krane, J. Dexamethasone and Dexamethasone Phosphate Detected
1
19
by H and F NMR Spectroscopy in the Aqueous Humour. Exp. Eye
Res. 1998, 66, 327−337.
7
(
0, 226−233.
6) Jimenez-Kairuz, A. F.; Allemandi, D. A.; Manzo, R. H. A strategy
(26) Avdeef, A.; Berger, C. M. pH-metric solubility. Dissolution
to improve the chemical stability of a model basic drug by ion pairing
with acid groups of polyelectrolytes. Int. J. Pharmaceutics 2004, 269,
titration template method for solubility determination. Eur. J. Pharm.
Sci. 2001, 14, 281−291.
1
(
49−156.
(27) In-vitro and in-vivo evaluation of dosage forms <1088>. United
7) Luppi, B.; Bigucci, F.; Cerchiara, T.; Zecchi, V. Chitosan-based
States Pharmacopoeia, The National Formulary and Dispensing
Information (USP 30-NF 25); USP: Rockville, MD, 2007.
hydrogels for nasal drug delivery: from inserts to nanoparticles. Expert
Opin. Drug Delivery 2010, 7, 811−828.
(
28) Menjoge, A. R.; Kulkarni, M. G. Mechanistic investigations of
phase behavior in Eudragit E blends. Int. J. Pharmaceutics 2007, 343,
06−121.
29) Caz
Pascual, A.; Ganem-Rondero, A.; Quintanar-Guerrero, D.; Lop
(
́
8) Kaminski, K.; Szczubiałka, K.; Zazakowny, K.; Lach, R.;
Nowakowska, M. Chitosan derivatives as novel potential heparin
1
reversal agents. J. Med. Chem. 2010, 53, 4141−4147.
(
́
ares-Delgadillo, J.; Balaguer-Fernan
́
dez, C.; Calatayud-
ez-
(
9) Vijay, P.; Taori, Y. L.; Theresa, M. R. DNA delivery in vitro via
́
surface release from multilayer assemblies with poly-
glycoamidoamine)s. Self-Assembling Biomater. 2009, 5, 925−933.
10) Esposito, E.; Sebben, S.; Cortesi, R.; Menegatti, E.; Nastruzzi, C.
Castellano, A. C.; Merino, V.; Kalia, Y. N. Transdermal iontophoresis
of dexamethasone sodium phosphate in vitro and in vivo: Effect of
experimental parameters and skin type on drug stability and transport
kinetics. Eur. J. Pharm. Biopharm. 2010, 75, 173−178.
(
(
Preparation and characterization of cationic microspheres for gene
delivery. Int. J. Pharmaceutics 1999, 189, 29−41.
(
30) Moustafine, R. I.; Zaharov, I. M.; Kemenova, V. A.
(
11) Lu, Z.-Z.; Wu, J.; Sun, T.-M.; Ji, J.; Yan, L.-F.; Wang, J.
Physicochemical characterization and drug release properties of
Eudragit E PO/Eudragitw L 100−55 interpolyelectrolyte complexes.
Eur. J. Pharm. Biopharm. 2006, 63, 26−36.
Biodegradable polycation and plasmid DNA multilayer film for
prolonged gene delivery to mouse osteoblasts. Biomaterials 2008, 29,
7
(
33−741.
(31) Moustafine, R. I.; Kemenova, V. A.; Van den Mooter, G.
12) Morille, M.; Passirani, C.; Vonarbourg, A.; Clavreul, A.; Benoit,
Characteristics of interpolyelectrolyte complexes of Eudragit E100
J. P. Progress in developing cationic vectors for non-viral systemic gene
with sodium alginate. Int. J. Pharmaceutics 2005, 294, 113−120.
therapy against cancer. Biomaterials 2008, 29, 3477−3496.
(32) Herlinger, A. W.; Ferraro, J. R.; Garcia, J. A.; Chiarizia, R. A far-
(
13) Quinteros, D. A.; Ramirez Rigo, M. V.; Jimenez Kairuz, A. F.;
infrared study and investigation of the difference between the
asymmetric and symmetric POO- stretching frequencies in metal-
diphosphonate complexes. Polyhedron 1998, 17, 1471−1475.
Olivera, M. E.; Manzo, R. H.; Allemandi, D. A. Interaction between a
cationic polymethacrylate (Eudragit E100) and anionic drugs. Eur. J.
Pharm. Sci. 2008, 33, 72−79.
(33) Baird, J. A.; Taylor, L. S. Evaluation of amorphous solid
(
14) Anwer, K; Rhee, B. G.; Mendiratta, S. K. Recent progress in
dispersion properties using thermal analysis techniques. Adv. Drug
Delivery Rev. 2012, 64, 396−421.
polymeric gene delivery systems. Crit. Rev. Ther. Drug Carrier Syst.
2
(
(
003, 20, 249−293.
(34) Siepmann, F.; Le Brun, V.; Siepmann, J. Drugs acting as
15) Akhtar, S. Beyond delivery. Gene Ther. 2006, 13, 739−740.
plasticizers in polymeric systems: a quantitative treatment. J. Controlled
Release 2006, 115, 298−306.
16) Rowe, R. C.; Sheskey, P. J.; Owen, S. C. Polymethacrylates. In
Handbook of Pharmaceutical Excipients, 5th ed.; American Pharma-
ceutical Association and Pharmaceutical Press: London, 2006; pp
(35) Goddeeris, C.; Willems, T.; Houthoofd, K.; Martens, J. A.; Van
den Mooter, G. Dissolution enhancement of the anti-HIV drug UC
81 by formulation in a ternary solid dispersion with TPGS 1000 and
EudragitE100. Eur. J. Pharm. Biopharm. 2008, 70, 861−868.
36) Weuts, I.; Kempen, D.; Verreck, G.; Peeters, J.; Brewster, M.;
5
(
25−533.
17) Raob, V. M.; Engha, K.; Qiu, Y. Design of pH-independent
controlled release matrix tablets for acidic drugs. Int. J. Pharmaceutics
003, 252, 81−86.
́
18) Pereira de Souza, T.; Martínez-Pacheco, R.; Gomez-Amoza, J.
7
(
2
(
Blaton, N.; Van den Mooter, G. Salt formation in solid dispersions
consisting of polyacrylic acid as a carrier and three basic model
compounds resulting in very high glass transition temperatures and
constant dissolution properties upon storage. Eur. J. Pharm. Sci. 2005,
25, 387−393.
L.; Petrovick, P. R. Eudragit E as Excipient for Production of Granules
and Tablets From Phyllanthus niruri L Spray-Dried Extract. AAPS
PharmSciTech 2007, 8, Article 34 (http://www.aapspharmscitech.org).
(
19) Hu, Y; Wu, Y. Y.; Xia, X. J.; Wu, Z.; Liang, W. Q.; Gao, J. Q.
(
37) Lee, H.-K.; Hajdu, J.; McGoff, P. Propranolol: methacrylic acid
copolymer binding interaction. J. Pharm. Sci. 1991, 80, 178−180.
38) Scheler, U. NMR on polyelectrolytes. Curr. Opin. Colloid
Development of drug-in-adhesive transdermal patch for α-asarone and
in vivo pharmacokinetics and efficacy evaluation. Drug Delivery 2011,
(
1
(
8, 84−9.
20) Mustafin, R. I. Interpolymer combinations of chemically
Interface Sci. 2009, 14, 212−215.
complementary grades of Eudragit copolymers: a new direction in
the design of peroral solid dosage forms of drug delivery systems with
controlled release. Pharm. Chem. J. 2011, 45, 285−295.
(39) Qiu, Y.; Park, K. Environment-sensitive hydrogels for drug
delivery. Adv. Drug Delivery Rev. 2001, 53, 321−339.
(40) Vilches, A. P.; Jimenez Kairuz, A. F.; Alovero, F.; Olivera, M. E.;
(
21) Boeris, V.; Romaninia, D.; Farruggi, B. Interaction and complex
Allemandi, D.; Manzo, R. H. Release Kinetics and up-take studies of
model fluoroquinolones from Carbomer hydrogels. Int. J. Pharmaceu-
tics 2002, 246, 17−24.
(41) Jimenez-Kairuz, A. F.; Allemandi, D. A.; Manzo, R. H.
Equilibrium properties and mechanism of kinetic release of
metoclopramide from carbomer hydrogels. Int. J. Pharmaceutics
2003, 250, 129−136.
formation between catalase and cationic polyelectrolytes: Chitosan and
Eudragit E100. Int. J. Biol. Macromol. 2009, 45, 103−108.
(
22) Haining, W. N.; Anderson, D. G.; Little, S. R.; von Berwelt-
Baildon, M. S.; Cardoso, A. A.; Alves, P.; Kosmatopoulos, K.; Nadler,
L. M.; Langer, R.; Kohane, D. S. pH-Triggered Microparticles for
Peptide Vaccination. J. Immunol. 2004, 173, 2578−2585.
2
432
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433

Molecular Pharmaceutics
Article
(
42) Wu, J.; Luan, M.; Zhao, J. Trypsin immobilization by direct
adsorption on metal ion chelated macroporous chitosan-silica gel
beads. Int. J. Biol. Macromol. 2006, 39, 185−191.
(
43) Hori, M.; Onishi, H.; Machida, Y. Evaluation of Eudragit-coated
chitosan microparticles as an oral immune delivery system. Int. J.
Pharmaceutics 2005, 297, 223−234.
2
433
dx.doi.org/10.1021/mp300282f | Mol. Pharmaceutics 2012, 9, 2424−2433