620 FARMACIA, 2010, Vol.

58, 5

PHASE SOLUBILITY STUDIES OF THE

INCLUSION COMPLEXES OF REPAGLINIDE
WITH β-CYCLODEXTRIN AND
β-CYCLODEXTRIN DERIVATIVES
CAMELIA NICOLESCU*, CORINA ARAMĂ, ANGELA NEDELCU,
CRINA-MARIA MONCIU
Analytical Chemistry Department, Faculty of Pharmacy, UMF Carol
Davila, Traian Vuia Street no. 6, Bucharest, Romania
*
corresponding author: nicolescu_camelia@yahoo.com

Abstract
The present study is intended to evaluate the possibilities to improve repaglinide
- an oral antidiabetic drug - solubility in water, based on inclusion complexes formation
with β-cyclodextrin (β-CD), hydroxypropyl β-cyclodextrin (HP-β-CD) and randomly
methylated β-cyclodextrin (RAMEB), respectively, and also to estimate their composition
and apparent stability constants, Kst, from the phase-solubility diagrams.
We have noticed that the phase solubility diagram for the repaglinide – HP-β-CD
inclusion complex is A type, while those of repaglinide - β-CD and RAMEB are B type.
Taking into account the lipophilicity of the zwitterionic structure of repaglinide, all
determinations were performed in buffered phosphate solutions (pH 6).
The phase-solubility diagrams indicate an enhancement of the repaglinide
solubility in the presence of β-CD, as well as its derivatives, HP-β-CD and RAMEB in
different extents, related to the type of cyclodextrin.

Rezumat
Studiul prezentat are ca scop evaluarea posibilităţilor de creştere a solubilităţii în
apă a repaglinidei (antidiabetic oral) prin intermediul formării unor complecşi de incluziune
cu β-ciclodextrina (β-CD), hidroxipropil β-ciclodextrina (HP-β-CD) şi β-ciclodextrina
metilată aleator (RAMEB) şi, de asemenea, determinarea, din diagramele de fază-
solubilitate, a rapoartelor de combinare şi a constantelor aparente de stabilitate, Kst.
Diagrama de fază-solubilitate în cazul complexului de incluziune repaglinidă –
HP-β-CD este de tip A, în timp în cazul complecşilor repaglinidei cu β-CD şi RAMEB,
diagramele de fază-solubilitate sunt de tipul B. Ţinând cont de lipofilia structurii amfionice
a repaglinidei, toate determinările au fost realizate în soluţii tampon fosfat (pH=6).
Diagramele de fază – solubilitate indică o creştere a solubilităţii repaglinidei în prezenţa β-
ciclodextrinei, precum şi a derivaţilor acesteia, HP-β-CD şi RAMEB, în proporţii care
variază în funcţie de tipul ciclodextrinei.

Keywords: repaglinide, inclusion complexes, β-cyclodextrin, hydroxypropyl-

β-cyclodextrin, randomly methylated β-cyclodextrin, phase solubility diagrams
FARMACIA, 2010, Vol.58, 5 621

Introduction
The ability of the cyclodextrins to form inclusion complexes with a
variety of organic compounds is based on their ability to provide a
hydrophobic cavity in aqueous solution for a hydrophobic guest molecule or
hydrophobic moieties in the guest molecule.
The importance of such inclusion complexes, widely used in the
pharmaceutical domain [1,2,4,13,14], is connected to the possibility to
improve the aqueous solubility of the drug substances, or to increase the
guest molecule stability, as well as the possibility to control drug release,
with many potential applications in drug formulations. The enhancement of
water solubility of drugs is especially important for their bioavailability [5].
Repaglinide, 2-ethoxy-4-[2-[[(1S)-3-methyl-1-[2-(1-piperidinyl)phenyl]
butyl]amino]-2-oxoethyl]benzoic acid is an oral antidiabetic drug
administered in patients with type 2 diabetes mellitus, with very low water
solubility (34 µg/mL at 37°C) and high lipophilicity (logP = 3.97) [7]. The
chemical structure of repaglinide is presented in Figure 1[10].

Figure 1.
Repaglinide chemical structure

We have investigated the ability of repaglinide to form inclusion

complexes with β-cyclodextrin (β-CD), hydroxypropyl β-cyclodextrin (HP-
β-CD) and randomly methylated β-cyclodextrin (RAMEB), in order to
evaluate their effect on repaglinide solubility in water. The synthesis and
characterization of these inclusion complexes are presented elsewhere [9].
The phase-solubility diagrams obtained according to the Higuchi and
Connors method [6] allowed us to estimate the molar ratio and the apparent
stability constants of the inclusion complexes formed.

Materials and methods

Repaglinide, with a purity of 97.69% was obtained from Novo
Nordisk A/S, Denmark. Beta-cyclodextrin was purchased from Cyclolab
(Hungary); 2- hydroxypropyl- β-cyclodextrin and the randomly methylated-
622 FARMACIA, 2010, Vol.58, 5

β cyclodextrin (substitution rate 1.7-1.8) were obtained from Fluka (Sigma -

Aldrich Chemie GmbH, Germany).
Phosphoric acid and sodium hydroxide used for the preparation of
the buffers were of analytical grade. Deionized ultrapure water for
chromatography was used as a solvent (prepared using a Barnstead
EasyPure RoDi apparatus)
10 mM phosphate buffer solutions were prepared using H3PO4 85%
solution and NaOH 1 M solution. The pH values (3.00; 4.00; 5.00; 6.00;
7.00 and 8.00) were adjusted under potentiometrical control, using a
Metrohm 716 DMS Titrino potentiometer.
For the solubility studies we used a Vortex -Genie®2 (Scientific
Industries) shaker and a Perkin-Elmer Lambda 2 UV/VIS Spectrometer.
The experiments were conducted in triplicate.
Excess amounts of drug (approx. 100 mg repaglinide) were added to
10 mL vials containing aqueous solutions of increasing concentrations of
cyclodextrins (10-3M – 10-1M for HP-β-CD and 2.5·10-3 M – 2·10-2M for β-
CD and RAMEB) and shaken at 25±0.5°C. In order to establish the optimal
mixing time, vials were shaken for 24 hours, respectively 72 hours. The
comparative results showed no significant differences. Hence, the shaking
time was set to 24 hours.
After 24 hours mixing, the samples were filtered through a 0.2 µm
Nylon filter membrane (Whatman® PuradiscTM) and the absorbance at λ 283
nm was measured, in order to determine the concentration of the dissolved
repaglinide.

Results and discussion

Repaglinide is an acid-base ampholyte, with two protonation sites
and thus with four possible species in equilibrium with each other. The pKa
values, determined by potentiometric titration are: pKa1 = 4.16 ± 0.06 and
pKa2 = 6.01 ± 0.04. At the isoelectric pH (5.50), two neutral forms of
repaglinide (zwitterionic and uncharged form) exist, resulting in a
significant lipophilicity of this molecule [7].
In order to establish the optimal pH value of the solution used for the
solubility studies, we have investigated the pH influence on the repaglinide
solubility in the presence and in absence of cyclodextrins.
The repaglinide concentration was calculated using the previously
determined specific absorbance (A1%1cm =58). Cyclodextrins do not interfere
in the measurement, as it can be seen in Figures 2 and 3.
FARMACIA, 2010, Vol.58, 5 623

Figure 2.
UV spectrum for repaglinide 0.5 mg/mL

Figure 3.
UV spectrum for HP- β -CD 0.1 M
As shown in Figure 4, the CDs influence on repaglinide solubility is
significant in the pH range 4-7, but with a pH higher than 6 a cumulative
effect of both CDs and pH occurs. It can be observed that repaglinide
minimum solubility is for pH values between 4 and 6. Our studies were
performed according to the method reported by Higuchi and Connors [6], in
buffered phosphate solutions, at a pH of 6, the region where the CDs effect
on solubility is predominant as the complexation is highly influenced by the
624 FARMACIA, 2010, Vol.58, 5

hydrophobicity of the repaglinide (also, the pH of the mutual mixture of

repaglinide and β- cyclodextrin and its derivatives is 5.9-6.2).

Figure 4.
pH and CDs influence on the water solubility of repaglinide

Phase-solubility diagrams
According to the Higuchi and Connors classification [6], the phase
solubility diagram for the repaglinide – HP- β-CD inclusion complex is A
type, which indicates the formation of a soluble complex. The shape of the
plot indicates that a different mechanism of solubilization is probably
involved for very low levels of HP-beta-CD (Figure 5). The complexation
may occur with a sufficient concentration of CD, higher than cca. 0.01 M
[1] Calculated regression parameters (slope = 10-5) allowed us to assume
that a 1:1 inclusion complex is mostly present in the aqueous solution.
In the case of the inclusion complexes between repaglinide and β-
CD or RAMEB respectively, the phase solubility diagrams are B type,
indicating an increase of solubility for repaglinide with the increase of the
cyclodextrin concentration up to approx. 1.5·10-2 M, followed by a plateau,
probably correlated with a limited solubility of the two complexes. (Figure 6
and Figure 7)
FARMACIA, 2010, Vol.58, 5 625

The shape of the solubility curve may indicate that a 1:1 molar ratio
is most probable for the inclusion complexes formed (slope = 6.8·10-3 for β-
CD and 6.4·10-3 for RAMEB) [9].
For the estimation of the apparent stability constants, Kst, we used
the parameters of the linear part of the solubility diagrams on the
assumption that 1:1 complexes were initially formed [8,11,12].
slope
According to the Higuchi-Connors equation: K st. = , the
S 0 (1 − slope )
calculated stability constants are:
Kst = 377.4 M-1 for repaglinide – HP- β-CD complex, Kst = 148.3 M-1 for
repaglinide – β-CD complex and Kst = 172.6 M-1 for repaglinide –RAMEB
complex .

Figure 5.
Phase solubility diagram for repaglinide- HP- β-CD complex
(Kst. = 377.4 M-1)

Figure 6.
Phase solubility diagram for repaglinide-β-CD complex (Kst. = 148.3 M-1)
626 FARMACIA, 2010, Vol.58, 5

Figure 7.
Phase solubility diagram for repaglinide- RAMEB complex
(Kst. = 172.6 M-1)

The most probable molecular model for the 1:1 complex repaglinide:
HP- β-CD is presented in Figure 8.

Figure 8.
Molecular modeling for the 1:1 complex repaglinide: HP- β-CD
(Hyperchem 6.0)

Conclusions
β-CD and its derivatives, HP-β-CD and RAMEB enhance the
solubility of repaglinide, a drug substance practically insoluble in water.
FARMACIA, 2010, Vol.58, 5 627

The CDs studied form 1:1 inclusion complexes with repaglinide. HP-β-CD
gives a soluble complex, while in the presence of β-CD and RAMEB,
inclusion complexes with limited solubility are formed.
According to the calculated apparent stability constants, the stability
of the complexes of repaglinide with β-CD, HP-β-CD and RAMEB varies
as follows: HP-β-CD > RAMEB > β-CD.

Acknowledgements

We thank Mr. Musa I. El-Barghouthi (Chemistry Department,

Faculty of Science, The Hashemite University, Zarka, Jordan) for the
valuable help in establishing the molecular model for the repaglinide: HP-
β-CD 1:1 inclusion complex, and Novo Nordisk A/S for the kind support of
providing the drug substance, repaglinide.

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Manuscript received: December 20th 2009