Acid-Catalyzed Hydrolysis of Ma1tosyl-pcyclodextrin FUMITOSHI HIRAYAMA, MASANOBU YAMAMOTO, AND KANETO UEKAMA' Received February 5, 1991, from the Faculty of Pharmaceutical Sciences, Kumamoto University, 5-7, Oe-honmachi, Kumamoto 862, Accepted for publicatron October 17, 1991.
Japan.
Abatract 0 Maltosyl-pcyclodextrinwas hydrolyzed via two pathways in acidic solution: (1) ring opening to give noncyclic oligosaccharides and (2) cleavage of maltose in the branched residue to give glucosyl-pCyD and glucose. Ring opening was -2-3 times faster than maltose cleavage because of the multiple hydrolysissitesof the pcyclodextrin (pCyD) ring (seven glycosidic linkages) compared with only one reaction site of the maltose residue in the branch. Values of the enthalpy and entropy of
activation of the hydrolyses were positive and in the range reported for maltose, a result indicating that the hydrolyses proceeded according to the A-1 mechanism ( i a , unimolecular decomposition). The a l , 6 glycosidic bond of branched pCyDs connecting pCyD and branched sugar moieties resisted hydrolysis; this property is a potential pharmaceutical advantage because the parent pCyD, which has low aqueous solubilii, would not precipitate after hydrolysis.
Branched cyclodextrins (CyDs) are highly hydrophilic derivatives that have sugar moieties substituted at primary hydroxyl groups via an a-1,Sglycosidic bond and that form inclusion complexes with various drug molecules.l.2 Branched CyDs are promising candidates as fast-dissolving carriers for poorly water soluble drugs, because their aqueous solubility 0 5 0 8 , w/v a t 25 "C) and biocompatibility are superior to those of the parent pCyD.M Reports on the chemical stability of branched CyDs are Bcarce.3 From a kinetic point of view, branched CyDs are of interest, because they have three types of glycosidic linkage that are susceptible to acid-catalyzed hydrolysis: (1)a-l,4-bonds in the CyD ring, (2) a-1,Sbonds at a junction between the ring and the branched sugars, and (3) a-1,Cbonds in the branched sugar moieties. In this study, the kinetics of the acid-catalyzed hydrolysis of maltosyl-pCyD (G,-pCyD; Scheme I) was investigated.
Experimental Section Material+PCyD was supplied by Nihon Shokuhin Kako Company (Tokyo, Japan) and was recrystallized from water. Highly purified G,-PCyD and glucosyl-PCyDs (Gl-pCyDs; >99.9%) were donated by Ensuiko Sugar Refinning Company (Yokoyama, Japan). All other materials and solvents were of analytical reagent grade. Deionized, double-distilled water was used. KineticgG,-&CyD (2.5 x LO-' M) was hydrolyzed in 0.1 M HCl-KCl buffer [ionic strength ( p ) = 0.2, pH 1.11 at various temperatures. At timed intervals, the reaction solution (0.5 mL) was sampled and neutralized with 0.1 M NaOH (0.5 mL) containing a-CyD (2.5 X lo-' M) as a n internal standard for analysis by high-performance liquid chromatography (HPLC). The neutralized solution (20 &) was analyzed by HPLC for simultaneous determination of Ga-PCyD, G1-PCyD, and parent PCyD. The HPLC system comisted of a pump (Hitachi 655A, Tokyo, Japan), a detector (Shodex SE-51 differential refractometer, Tokyo, Japan), a column (ERC-NH1181,6 mm diameter x 250 mm; Erma Optical Works, Tokyo, Japan), a mobile phase of acetonitri1e:water (65:35, vlv), and a flow rate of 1.2 mumin. The three PCyDs were well separated (Figure l),eJ and linear calibration curves were obtained over the concentration range (1.0 x 10-'-2.5 x lo-' M) of PCyDs. The following data were obtained from plots of concentrations of pCyD, GI-pCyD, and G,-PCyD, respectively, versus peak height ratios (PCyDs:internal GV~-3~9/92/0900-O913$02..50/0 0 1992, American Pharmaceutical Association
non-cyclicoligosaccharides
k5
Schemo I-Proposed hydrolysis pathways of G,-pCyD. Key: (a) glucose unit; (U a-l,4-glycosidic ) linkage; (-a) a-l,6-
glycosidic linkage.
standard): slopes of 7.244 x lo-', 8.343 x LO-', and 8.951 x lo-'; intercepts of 9.980 x loTs, 2.297 x lo-', and 1.300 x and correlation coefficients of 0.995,0.991,and 0.993. Rate constants were determined from an average of three measurements, which were within 5%of each other.
Results and Discussion Glycosides may be degraded by hydrolysis, epimerization, and dehydration. Among these reactions, acid-catalyzed hydrolysis of glycosidic linkages predominates in dilute solutions of substrates at temperatures
Japan.
Abatract 0 Maltosyl-pcyclodextrinwas hydrolyzed via two pathways in acidic solution: (1) ring opening to give noncyclic oligosaccharides and (2) cleavage of maltose in the branched residue to give glucosyl-pCyD and glucose. Ring opening was -2-3 times faster than maltose cleavage because of the multiple hydrolysissitesof the pcyclodextrin (pCyD) ring (seven glycosidic linkages) compared with only one reaction site of the maltose residue in the branch. Values of the enthalpy and entropy of
activation of the hydrolyses were positive and in the range reported for maltose, a result indicating that the hydrolyses proceeded according to the A-1 mechanism ( i a , unimolecular decomposition). The a l , 6 glycosidic bond of branched pCyDs connecting pCyD and branched sugar moieties resisted hydrolysis; this property is a potential pharmaceutical advantage because the parent pCyD, which has low aqueous solubilii, would not precipitate after hydrolysis.
Branched cyclodextrins (CyDs) are highly hydrophilic derivatives that have sugar moieties substituted at primary hydroxyl groups via an a-1,Sglycosidic bond and that form inclusion complexes with various drug molecules.l.2 Branched CyDs are promising candidates as fast-dissolving carriers for poorly water soluble drugs, because their aqueous solubility 0 5 0 8 , w/v a t 25 "C) and biocompatibility are superior to those of the parent pCyD.M Reports on the chemical stability of branched CyDs are Bcarce.3 From a kinetic point of view, branched CyDs are of interest, because they have three types of glycosidic linkage that are susceptible to acid-catalyzed hydrolysis: (1)a-l,4-bonds in the CyD ring, (2) a-1,Sbonds at a junction between the ring and the branched sugars, and (3) a-1,Cbonds in the branched sugar moieties. In this study, the kinetics of the acid-catalyzed hydrolysis of maltosyl-pCyD (G,-pCyD; Scheme I) was investigated.
Experimental Section Material+PCyD was supplied by Nihon Shokuhin Kako Company (Tokyo, Japan) and was recrystallized from water. Highly purified G,-PCyD and glucosyl-PCyDs (Gl-pCyDs; >99.9%) were donated by Ensuiko Sugar Refinning Company (Yokoyama, Japan). All other materials and solvents were of analytical reagent grade. Deionized, double-distilled water was used. KineticgG,-&CyD (2.5 x LO-' M) was hydrolyzed in 0.1 M HCl-KCl buffer [ionic strength ( p ) = 0.2, pH 1.11 at various temperatures. At timed intervals, the reaction solution (0.5 mL) was sampled and neutralized with 0.1 M NaOH (0.5 mL) containing a-CyD (2.5 X lo-' M) as a n internal standard for analysis by high-performance liquid chromatography (HPLC). The neutralized solution (20 &) was analyzed by HPLC for simultaneous determination of Ga-PCyD, G1-PCyD, and parent PCyD. The HPLC system comisted of a pump (Hitachi 655A, Tokyo, Japan), a detector (Shodex SE-51 differential refractometer, Tokyo, Japan), a column (ERC-NH1181,6 mm diameter x 250 mm; Erma Optical Works, Tokyo, Japan), a mobile phase of acetonitri1e:water (65:35, vlv), and a flow rate of 1.2 mumin. The three PCyDs were well separated (Figure l),eJ and linear calibration curves were obtained over the concentration range (1.0 x 10-'-2.5 x lo-' M) of PCyDs. The following data were obtained from plots of concentrations of pCyD, GI-pCyD, and G,-PCyD, respectively, versus peak height ratios (PCyDs:internal GV~-3~9/92/0900-O913$02..50/0 0 1992, American Pharmaceutical Association
non-cyclicoligosaccharides
k5
Schemo I-Proposed hydrolysis pathways of G,-pCyD. Key: (a) glucose unit; (U a-l,4-glycosidic ) linkage; (-a) a-l,6-
glycosidic linkage.
standard): slopes of 7.244 x lo-', 8.343 x LO-', and 8.951 x lo-'; intercepts of 9.980 x loTs, 2.297 x lo-', and 1.300 x and correlation coefficients of 0.995,0.991,and 0.993. Rate constants were determined from an average of three measurements, which were within 5%of each other.
Results and Discussion Glycosides may be degraded by hydrolysis, epimerization, and dehydration. Among these reactions, acid-catalyzed hydrolysis of glycosidic linkages predominates in dilute solutions of substrates at temperatures
