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  • FARMACIA, 2010, Vol.58, 5



    Analytical Chemistry Department, Faculty of Pharmacy, UMF Carol Davila, Traian Vuia Street no. 6, Bucharest, Romania *corresponding author: [email protected]


    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 -cyclodextrin (-CD), hydroxypropyl -cyclodextrin (HP--CD) and randomly methylated -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--CD inclusion complex is A type, while those of repaglinide - -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 -CD, as well as its derivatives, HP--CD and RAMEB in different extents, related to the type of cyclodextrin.


    Studiul prezentat are ca scop evaluarea posibilitilor de cretere a solubilitii n ap a repaglinidei (antidiabetic oral) prin intermediul formrii unor compleci de incluziune cu -ciclodextrina (-CD), hidroxipropil -ciclodextrina (HP--CD) i -ciclodextrina metilat aleator (RAMEB) i, de asemenea, determinarea, din diagramele de faz-solubilitate, a rapoartelor de combinare i a constantelor aparente de stabilitate, Kst.

    Diagrama de faz-solubilitate n cazul complexului de incluziune repaglinid HP--CD este de tip A, n timp n cazul complecilor repaglinidei cu -CD i RAMEB, diagramele de faz-solubilitate sunt de tipul B. innd cont de lipofilia structurii amfionice a repaglinidei, toate determinrile au fost realizate n soluii tampon fosfat (pH=6). Diagramele de faz solubilitate indic o cretere a solubilitii repaglinidei n prezena -ciclodextrinei, precum i a derivailor acesteia, HP--CD i RAMEB, n proporii care variaz n funcie de tipul ciclodextrinei.

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

    -cyclodextrin, randomly methylated -cyclodextrin, phase solubility diagrams

  • FARMACIA, 2010, Vol.58, 5



    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 g/mL at 37C) 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 -cyclodextrin (-CD), hydroxypropyl -cyclodextrin (HP--CD) and randomly methylated -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- -cyclodextrin and the randomly methylated-

  • FARMACIA, 2010, Vol.58, 5


    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 -Genie2 (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--CD and 2.510-3 M 210-2M for -CD and RAMEB) and shaken at 250.5C. 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 m Nylon filter membrane (Whatman PuradiscTM) and the absorbance at 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


    Figure 2.

    UV spectrum for repaglinide 0.5 mg/mL

    Figure 3.

    UV spectrum for HP- -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

  • FARMACIA, 2010, Vol.58, 5


    hydrophobicity of the repaglinide (also, the pH of the mutual mixture of repaglinide and - 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- -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 -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. 1.510-2 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


    The shape of the solubility curve may indicate that a 1:1 molar ratio is most probable for the inclusion complexes formed (slope = 6.810-3 for -CD and 6.410-3 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].

    According to the Higuchi-Connors equation: ( )slope10Sslope

    st.K = , the

    calculated stability constants are: Kst = 377.4 M-1 for repaglinide HP- -CD complex, Kst = 148.3 M-1 for repaglinide -CD complex and Kst = 172.6 M-1 for repaglinide RAMEB complex .

    Figure 5.

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

    Figure 6.

    Phase solubility diagram for repaglinide--CD complex (Kst. = 148.3 M-1)

  • FARMACIA, 2010, Vol.58, 5


    Figure 7.

    Phase solubility diagram for repaglinide- RAMEB co