STUDIA UNIVERSITATIS BABEŞ-BOLYAI, PHYSICA, SPECIAL ISSUE, 2003
BIMODAL MOLECULAR ENCAPSULATION OF MEFENAMIC ACID BY
β-CD IN SOLUTION AND SOLID STATE
M. Bogdan1, Diana Bogdan1, M.R. Caira2, S.I. Fărcaş1
1
National Institute for Research and Development of
Isotopic and Molecular Technologies, P.O. Box 700,
Donath Str. # 71-103, 400293 Cluj-Napoca 5,
Romania
Tel. +4-0264-584037, Fax: +4-0264-420042
2
University of Cape Town, Department of
Chemistry, Rondebosch 7701, South Africa
Tel. 021 6503071, Fax. 021 6897499
E-mail: xraymino@science.uct.ac.za
Abstract. Inclusion complexes of mefenamic acid anion (MF–), a nonsteroidal anti-inflammatory drug, with β-cyclodextrin (β-CD) were
prepared and characterized both in solution and in solid state using 1H
NMR and X-ray diffraction studies. Inclusion of the mefenamic acid
anion in the host molecule is shown by changes in the chemical shifts of
some of the guest and host protons, in comparison with the chemical
shifts of the same protons in the free compounds. The continuous
variation method was used to establish the stoichiometry. The obtained
results indicate that simultaneous inclusion of both rings occur, giving
rise to two isomeric 1:1 complexes. The association constants for the two
1:1 complexes were calculated by a non-linear least-squares regression
analysis of the changes in observed chemical shifts of the drug and β-CD
lines as a function of β-CD concentration. The obtained results were
compared with those obtained by X-ray powder diffraction in
combination with molecular mechanics calculations. The geometry of the
two 1:1 complexes according to the obtained X-ray data is given.
Introduction
Mefenamic acid is a non-steroidal drug with strong analgesic antiinflammatory and anti-pyretic properties, widely applied in therapeutics. The
therapeutic single oral dose of mefenamic acid is 0.25 g; it is absorbed from the
alimentary canal and after 2 hours the maximal concentration in the blood is
reached. Unfortunately, it may produce a number of side effects such as nausea,
vomiting, bleeding from the alimentary canal, rash, etc. The side effects can be
reduced by increasing the drug solubility, which enhances its biological availability
and permits decrease of the required dose. Moreover, this drug is not stable and
products of its decomposition can enhance undesirable effects. In the modern
technology of drug formulation, cyclodextrins are used as stability and solubilising
agents [1].
M. BOGDAN, DIANA BOGDAN, M.R. CAIRA, S. FĂRCAŞ
Cyclodextrins (CDs) have homogeneous toroidal structures of different
molecular size: most typical are cyclohexaamylose (α-CD), cycloheptaamylose (βCD) and cyclooctaamylose (γ-CD). All glucose units are slightly tilted so they
form a hollow truncated cone. The primary hydroxyl groups are located at the
wider rim and the secondary hydroxyl groups are found at the narrower rim. The
torroidal structure has a hydrophilic surface, making them water soluble, whereas
the cavity is composed of the glycoside oxygens and methine hydrogens, giving it a
hydrophobic character. As a consequence, the CDs are capable of forming
inclusion complexes with compounds having a size compatible with dimensions of
their size [2].
In a thoroughly study of an inclusion complex, three main points need to
be clarified: stoichiometry, association constant and geometry of the complexes.
Such a complete characterization of the inclusion complexes should contribute to a
better understanding of the therapeutic properties.
In this work, we investigated the complexation of mefenamic acid anion 1
(MF–) and β-CD, 2, in water by NMR and showed that two 1:1 complexes coexist.
The structure of these complexes in solid state was estimated from the X-ray
diffraction data in combination with molecular mechanics calculations.
Materials and Methods
β-cyclodextrin (β-CD) containing an average of 8 water molecule/
molecule was purchased from Sigma Chemie GmbH (Germany). The β-CD was
used without further purification and the water content was considered in the
calculation of solute concentrations. The D2O (deuterium content 99,7 %) was
purchased from Institute for Cryogenics and Isotope Separations (Rm. Vâlcea,
Romania).
a
COOH
b
h
f
c
NH
e
d
1
CH
3
1
CH
3
2
2
BIMODAL MOLECULAR ENCAPSULATION OF MEFENAMIC ACID BY Β-CD
The NMR experiments were performed at 300 MHz with a Varian-Gemini
spectrometer. The 1H NMR spectra were recorded in D2O solution at 293±0.5 K
and all chemical shifts were measured relative to external TMS. Typical conditions
were as follows: 16 K data points, sweep width 4500 Hz, giving a digital resolution
of 0.28 Hz/point. The 90˚ pulse width was 13 μs and the spectra were collected by
co-addition of 32 or 64 scans. In some cases, an appropriate Gaussian function was
applied before Fourier transformation to enhance the spectral resolution.
In order to study the complexation process between mefenamic acid (MF)
and β-CD in solution, two stock solutions in D2O, both having 10 mM were
prepared. Due to extremely low solubility of mefenamic acid in water [3], it was
converted to its sodium salt by titration with NaOD to a final pH = 12. Based on
these two equimolar solutions, a series of nine samples (i = 1÷9) containing both
the MF– and the β-CD molecules were prepared. This was accomplished by mixing
the two solution to constant volume (2 ml) at varying proportions, so that a
complete range (0 < r < 1) of the ratio r=[X]/([H] + [G]) was sampled. X = G or H
and [H] and [G] are the total concentrations of the host (β-CD) and guest (MF–),
respectively. Thus, the total concentration [H] + [G] = [M] = 10 mM was kept
constant for each solution. The same set of samples was used both for the
determination of the stoichiometry and association constant, Ka.
For X-ray diffraction study, the powder sample was prepared as follows.
Equal amounts (25 ml) of β-CD and MF-Na solutions were mixed, shaken for 8 h
and then stored for 7 days at 313 K. The precipitate of the inclusion complex was
washed twice with small portions of distilled water and was dried first in air and
then in a dryer at 325 K. A capillary of diameter 0.7 mm was filled with powder
and measured at the high-resolution powder station of BM16 at the European
Synchrotron Radiation Facility (Grenoble) with λ = 0.80081 Å. Continuous scans
were made from 0.0˚ to 48.0˚ in 2θ with 0.5˚ 2θ min and a sampling time of 50 ms.
Results and Discussion
Determination of the stoichiometry
Several techniques like IR, CD and UV-VIS spectroscopy can establish if
guest molecules form an inclusion complex with β-CD, but they cannot provide
information about the structural configuration of the complex. In contrast, NMR is
a technique which provides the most evidence for the inclusion of a guest into the
hydrophobic CD cavity in solution. Inclusion of MF– in β-CD is shown by the
change in the chemical shift of some of the guest and host protons in comparison
with the chemical shifts of the same protons in the free components. Partial 1H
NMR spectra of pure components and MF–:β-CD mixture in a 3:2 molar ratio are
shown in Figures 1 and 2.
The absence of new peaks that could be assigned to the complex suggested
that complexation is a dynamic process, the included MF– being in a fast exchange
between the free and bound states.
Determination of the stoichiometry of the MF–:β-CD complex by
continuous variation method was based on 1H NMR spectra obtained for MF– and
M. BOGDAN, DIANA BOGDAN, M.R. CAIRA, S. FĂRCAŞ
Figure 1. Partial 300 MHz spectra of (a) 10 mM β-CD and (b) 4 mM β-CD and 6 mM
mefenamic acid anion.
Figure 2. Partial 300 MHz spectra of (a) 10 mM mefenamic acid anion and (b) 6 mM
mefenamic acid anion and 4 mM β-CD. Only the spectral region for aromatic
protons (Ha, Hc, Hh, Hf, He, Hd, Hb) of mefenamic acid anion is displayed.
BIMODAL MOLECULAR ENCAPSULATION OF MEFENAMIC ACID BY Β-CD
β-CD mixtures in which the initial concentrations of the two species were
maintained constant and the ratio r varied between 0 and 1. The continuous
variation plots (Job plots) of |Δδ|·[β-CD] against r1 = m/(m + n), where m and n
are, respectively, the proportions of β-CD and MF-Na in the (MF-Na)n : (β-CD)m
complex are presented in Figure 3. The induced shift, Δδ, is defined as the
difference in chemical shifts in the absence and in the presence of the other reactant
for a given ratio r.
Figure 3. Job plots for protons H3, H5, and H6 of β-CD in the presence of
different concentrations of mefenamic acid anion
Thus, for H3, H5 and H6 protons of β-CD, significant upfield shifts,
attributable to the inclusion of an aromatic part, were observed. The Jobs plots
show a maximum at r1 = 0.5 and quite symmetrical shapes indicating that the
complex has 1:1 stoichiometry.
The MF-Na protons can be split into two groups, one shifted upfield (Ha,
He, Hh and H2) and the other (Hd) downfield. Because the protons belonging to
both the aromatic rings of MF-Na show chemical shift differences upon inclusion,
suggests that multiple equilibria may exist in solution. Although the shapes of the
Job plots for MF-Na protons are not smoothly and highly symmetrical, the
maximum does not deviate significantly from r2 = 0.5, indicating, in our opinion,
the existence of two isomeric 1:1 complexes. Similar behavior was reported for
other non-steroidal anti-inflammatory agents such as diclofenac [4], piroxicam [5],
naproxen [6] and an antiacetylcoline drug, oxyphenonium bromide [7].
M. BOGDAN, DIANA BOGDAN, M.R. CAIRA, S. FĂRCAŞ
Evaluation of the binding constants
In order to determine the extent of the intermolecular binding between the
two aromatic rings of MF-Na and β-CD, the association constants have been
evaluated.
The association constant, Ka, for a 1:1 complex can be determined
according to the following equation [8]:
1
2
2
j  







1
1
i 
i 
i , j 
c
  4H  G   


  M 
M 
2X  
K a 
Ka 
 


(1)
were i counts the sample number and j the investigated protons.
If the studied proton belongs to the guest or host molecule, then X = G or
H, respectively. Δδc(i) represents the chemical shift difference (for a given proton)
between the free component and a pure inclusion complex.
We developed a computer programme based on an iteration procedure
following specific algorithms in order to fit the experimental values Δδc(i,j) to the
appropriate equation. Each iteration sets up a quadratic programme to determine
the direction of search and the loss function:
i , j  2
(2)
E
 i , j    calc
 
i

j
until search converges. The fitting procedure reaches an end when the difference
between two consecutive E values is smaller than 10-6. The treatment of the whole
set of protons studied yields one single Ka value for the whole process and a set of
calculated δc(i,j) values.
In our particular case, we applied eq. (1) for a set of protons consisting in
H3, H5 and H6 of β-CD and Ha and Hd of MF-Na and then for H3, H5 and H6 of
β-CD and He, Hh and H2 of MF-Na. This means that we considered first the case
when the xylyl moiety is inserted in the β-CD cavity and then the inclusion of
benzoic acid moiety. The association constants obtained, using the above described
procedure are:
K1:1’ = 172.32 M-1
E = 3.11 · 10-3
r = 0.993
K1:1 = 435.54 M-1
E = 2.14 · 10-3
r= 0.995
Based on the observed chemical shift changes of H3,
H5, H6, Ha and Hd
Based on the observed chemical shift changes of H3,
H5, H6, He, Hh and H2
It is worth mentioning that there is a striking similarity between the K a
value obtained by us and the values reported by Ikeda et al. [9] using CD
(Ka = 620 M-1), UV-VIS spectroscopy (Ka = 630 M-1) and solubility measurements
(Ka = 570 M-1).
BIMODAL MOLECULAR ENCAPSULATION OF MEFENAMIC ACID BY Β-CD
Structure determination in solid state
The crystal structure of the inclusion complex of β-CD with MF-Na has
been determined from a combination of high-resolution synchrotron powder
diffraction data and molecular mechanics calculations [10]. A grid search indicates
two possible solutions, which are corroborated by molecular mechanics
calculations, while Rietveld refinement (RR) results suggest the crystal structure
that is more likely to be formed in the solid state. Thus, MF-Na is partially
included in β-CD with either the xylyl or the benzoic acid moiety being inside its
cavity.
After energy minimization, the two models presented in Figure 5 (to be
referred as I and II) were found to be almost isoenergetic and MF-Na had become
only partially encapsulated in the CD macrocycle. Either the xylyl (I) or the
benzoic acid (II) moiety was inside the cavity and the N atom that linked the two
phenyl rings was found at the secondary face of the macrocycle in both cases. After
RR was performed, a significantly better fit was obtained for (I), suggesting that in
the solid state, the solution that has the xylyl moiety partially included in the CD
cavity is more likely to be formed than solution (II). In our calculations, water
molecules were not considered because of the lack of stoichiometric information.
I
II
Figure 5. The partial inclusion of xylyl moiety (I) and benzoic acid moiety (II) in
the β-CD cavity
After convergence, the calculated solvent-accessible areas for (I) allow for
the presence of one water molecule inside CD cavity and of five more in the space
between the CD molecules while for (II) there is only space for about nine water
molecules outside the CD cavity. We can conclude than that in solid state both
models (I and II) MF-Na and β-CD form a monomeric complex (occupancy factor
0.9) in a herringbone-packing scheme in which CD faces are blocked by adjacent
CD molecules.
Conclusions
The mefenamic acid sodium salt : β-cyclodextrin inclusion complex has
been studied in aqueous solution by 1H NMR and in solid state by high-resolution
synchrotron powder diffraction technique and molecular mechanics calculations.
The induced chemical shifts in the NMR spectra prove the existence of a bimodal
M. BOGDAN, DIANA BOGDAN, M.R. CAIRA, S. FĂRCAŞ
binding between MF-Na and β-CD and give values for the binding constants. The
coexistence of the two 1:1 complexes also in solid state is confirmed by highresolution powder diffraction data. The crystal structure that is more likely to be
found in the solid state is suggested by RR agrees with the solution furnished by 1H
NMR.
Acknowledgements
The research was supported by the Romanian Ministry of Education,
Research and Youth and the BIOTECH programme (Project 01-8-CPD-041).
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