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351
the more pronounced change in chemical shift values of drug H9
protons in presence of EPI-b-CD and SBE-b-CD, compared to b-CD
(Table 2).
CD allowed deeper penetration of the drug into the central cavity
of this cyclodextrin derivate. This directly contributed to the for-
mation of a more stabile inclusion complex, as observed by phase
solubility studies.
The nOe cross-peaks observed in 2D ROESY spectra of BVP/SBE-
b-CD binary system belonged to SBE-b-CD intramolecular interac-
tions (Figure 3F and G) and to interaction between cyclodextrin
and BVP HCl (Figure 3G and H) The obtained results were a basis
for elucidation of the mechanism that contribute to the stability
of the BVP HCl/SBE-b-CD inclusion complex.
As in case of EPI-b-CD, chemical modification of SBE-b-CD re-
sulted in a distortion of the cone structure with respect to native
b-CD, allowing intramolecular interactions between H1 and other
protons of the cone (Figure 3F). Other intramolecular nOe cross
peaks were attributed to the interaction between protons of sul-
phobutylether sidearms and protons of the cone (Figure 3G). In
fact, sulphobutylether sidearms are capable of free rotation that al-
lowed their interaction with some protons of the cone, resulting in
strong nOe intramolecular cross-peaks. The interaction seems to be
more intense for middle ACH2ACH2A protons of sulphobutylether
sidearms of SBE-b-CD than for ACH2A group directly bonded to the
ASO3 group. The observed structural change of SBE-b-CD with re-
spect to parent b-CD seems to be more favourable for the complex-
ation of BVP HCl. Pronounced intermolecular nOe cross-peaks
indicated a strong interaction between cyclodextrin cone protons
and H11 and H12,13 protons of drug (Figure 3G and H). This con-
firmed that a complex between BVP HCl and SBE-b-CD has been
formed by inclusion of the methylated phenyl group of BVP HCl
into the central cyclodextrin cavity. The extension of its lipophilic
cavity caused by the presence of sulphobutylether sidearms on the
b-CD core contributed to more intense interactions between the
drug and SBE-b-CD, resulting in formation of the most stable inclu-
sion complex, as observed by phase solubility studies.
Moreover, the upfield shift of 1H HMR signals corresponding to
the sulphobutylether chains of SBE-b-CD confirmed their interac-
tion with the drug molecule. The signal of –CH2- group attached
to ASO3 group (H10,100, d = 2.834 ppm) showed the most pro-
nounced upfield shift in the drug presence (
while the signal corresponding to the middle ACH2CH2A protons
(H9,90 and H8,80, d = 1.661 ppm) was less affected (
D
d = ꢁ0.010 ppm),
D
d = ꢁ0.006
ppm). The interaction of BVP HCl with the sulphobutylether chains
of SBE-b-CD is mainly attributable to electrostatic attraction be-
tween the oppositely charged molecules. This interaction addition-
ally contributes to enhance the complex stability [25], leading to
the highest increase of the drug aqueous solubility (Figure 1).
4.3. Two dimensional 1H NMR studies (ROESY)
Two dimensional NMR is a powerful technique for investigation
of inter- and intra-molecular interactions. The presence of nOe
cross-peaks between protons of two different species in 2D ROESY
spectrum is an indication that they are in spatial contact through
space within 3–5 Å. Based on the results obtained from 2D ROESY
spectra, the spatial conformations of inclusion complexes may be
determined. Thus, to obtain more insight about the binding mode
between BVP HCl and cyclodextrins tested and to confirm the pro-
posed structure of inclusion complexes formed, 2D ROESY NMR
experiments were performed.
In 2D ROESY spectrum of BVP HCl/b-CD binary system, two
groups of intermolecular nOe cross-peaks were observed (Figure
3A and B): the first belongs to the interaction between H11 protons
of BVP HCl and b-CD protons, the other to the interactions between
H12 and 13 and b-CD protons. In both cases, interaction with inter-
nal and external b-CD protons was observed. One has to be aware
that an inclusion complex is characterized by existence of a dy-
namic equilibrium between the complexed and free drug forms.
This equilibrium is extremely fast, even on a NMR-timescale. Thus,
it is possible to detect nOe cross-peaks that are the consequence of
inclusion complex formation, as well as those that corresponded to
non-inclusion interactions between free drug and cyclodextrin
molecule in the solution. Moreover, cross-peaks observed between
BVP HCl protons and internal b-CD protons (H3 and H5) were al-
ways more intense than those of external b-CD protons (H2 and
H4), which may be taken as an indication that the inclusion com-
plex formation was the prevalent interaction between the compo-
nents. These results also confirmed the assumed structure of the
complex formed. The nOe cross-peak corresponding to the interac-
tion of the drug H9 protons and b-CD protons was not observed,
indicating that these protons are in greater distance, probably as
a consequence of the geometry of the inclusion complex formed.
The 2D ROESY spectrum of BVP/EPI-b-CD binary system showed
a strong intramolecular nOe cross-peak that may be attributed to
the interaction of H1 proton with other protons of CD core (Figure
3C). The same interaction was not observed in 2D ROESY spectrum
of parent b-CD. This result suggested that chemical modification of
b-CD has resulted in a distortion of the cone structure. As a conse-
quence, the binding mode between BVP HCl and EPI-b-CD has been
changed. Cross-peaks corresponding to the interaction of cyclodex-
trin protons with protons H11 and H12,13 of the drug molecule
were observed (d = 1.179–1.297 ppm and 3.031–3.3133 ppm, Fig-
ure 3D), but their intensity was reduced in comparison with those
of b-CD. Also, strong nOe cross-peaks corresponding to the interac-
tion between protons of piperidine ring of BVP HCl and EPI-b-CD
protons were observed (Figure 3E). This interaction was absent in
ROESY spectra of the sample with b-CD. Probably, the distortion
of the CD cone caused by chemical modification in case of EPI-b-
4.4. Molecular modeling studies
2D-ROESY spectra demonstrated several intermolecular cross-
peaks related to the nOe interactions between the internal protons
of the cyclodextrins (H3 and H5) and the aromatic and methyl pro-
tons of BVP HCl. Since the nOe cross-peak’s intensity values are in-
versely proportional to the sixth power of the distance between the
protons (nOe 1 1/r6), they can be correlated with the lowest mea-
surable distance between interacting protons. By using a known
distance as a reference (in this case that between the aliphatic pro-
tons H-8,80 and H6,60 of BVP HCl), it is possible to calculate the dis-
tances between interacting protons from the corresponding cross-
peaks intensity values according to the following equation:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
gref
gi;j
6
ri;j
¼
ꢀ r6ref
ð6Þ
where
ter-proton distance of two interacting protons i and j, respectively,
while ref and rref are the corresponding values of the reference pro-
gi,j and ri,j represent the nOe cross-peak intensity and the in-
g
tons. The mean values of the calculated distances for the BVP HCl
complexes with b-CD and SBE-b-CD are presented in Table 3. These
data were taken as a basis for the molecular dynamic calculations,
in order to hypothesize the most probable conformations of the
inclusion complexes formed.
Since BVP molecule has a chiral centre, 4 possible inclusion
modes have been considered in case of b-CD complex. In particular,
for each enantiomer, S and R, two different orientation modes into
the b-CD cavity have been considered, i.e. one with the aromatic
portion inserted from the secondary rim (A type complex) and an-
other from the primary rim (B type complex) of the cone.