- Open Access
Inclusion complexes of trihexyphenidyl with natural and modified cyclodextrins
© Maeda et al.; licensee Springer. 2015
- Received: 3 February 2015
- Accepted: 17 April 2015
- Published: 7 May 2015
The solubility of trihexyphenidyl (Thp) was improved by its combination with β-cyclodextrin (β−CD) and modified β-CDs. The solubility of Thp was found to be increased in the presence of β-CD, hydroxypropyl-β-cyclodextrin (HP-β-CD), methyl-β-cyclodextrin (Me-β-CD) and sulfobutylether-β-cyclodextrin (SBE-β-CD). In particular, the solubility of Thp in conjunction with SBE-β-CD was increased by a factor of 11. The formation constant (K c ) for the Thp/SBE-β-CD inclusion complex was determined to be 2300 L/mol based on fluorometry data. The structure of the Thp/SBE-β-CD complex in aqueous solution was examined by 1H-1H rotating frame nuclear Overhauser effect spectroscopy (ROESY) NMR, and the phenyl moiety of the Thp was found to coordinate with the secondary hydroxyl face of the SBE-β-CD. A solid Thp/SBE-β-CD inclusion complex was prepared by freeze-drying.
- Inclusion complexes
Trihexyphenidyl (Thp) is a pharmaceutical compound that has been shown to improve various disease symptoms, including muscle rigidity, finger tremors and depression, by regulating the release of adrenaline.
Natural cyclodextrins (α-, β- and γ-CD) are widely used in many fields since they readily form inclusion complexes with a variety of organic compounds (Saenger 1980; ÖZdemir and Ordu 1998; Reineccius et al. 2002). As an example, β-cyclodextrin (β-CD) is used to suppress the bitterness of antihistamine drugs in solution through the formation of inclusion complexes (Hibi et al. 1984; Ono et al. 2011). However, β-CD exhibits relatively low solubility in water, which limits its applications in pharmaceutical formulations. Therefore, various CD derivatives have been synthesized to extend the potential applications (Uekama 1985; Szejtli 1992; Loftsson and Duchêne 2007). We have previously reported complexes of α-lipoic acid and melatonin with modified CD derivatives (Maeda et al. 2010, 2013), and determined that the solubilities of α-lipoic acid and melatonin in the presence of sulfobutylether-β-CD increased by factors of 20 and 10, respectively.
In the present work, we investigated the solubility of inclusion complexes of Thp with natural and modified CDs at constant pH. To date, Thp complexes have not been assessed spectroscopically because of the low solubility of Thp. Haiyun et al. reported the evaluation of the complexation of rutin and β-CD by fluorescence spectroscopy in a phosphate solution containing 2% (v/v) methanol (Haiyun et al. 2003). Recently, Alvarez-Parrilla et al. reported stability constants for quercetin and rutin according to the methodology described by Haiyun (Alvarez-Parrilla et al. 2005). More recently, Al-Rawashdeh et al. reported the complexation of the sunscreen agents (oxybenzene, E-2-ethylhexyl-methoxycinnamate, octocrylene) and β-CD by UV-Vis spectroscopy in methanol/water mixture. The results demonstrate that the formation of inclusion complexes of the sunscreen agents and β-CD can inhibit the photodegradation (Al-Rawashdeh et al. 2010, 2013). Therefore, this method using an organic solvent was used in the present study to allow determination of the formation constants (K c) of Thp with various CDs.
The enantiodiscrimination of chiral Thp enantiomers by carboxylated methyls of α-, β-, and γ-CD was predicted by molecular docking study (Mulisa et al. 2014). More recently, structure of Thp/β-CD was elucidated by 1H NMR spectroscopic and computational methods (Ali and Shamim 2014).
The stability constant (K) and K c values of Thp with various CDs were obtained in a 50 mmol pH 7 phosphate buffer containing ethanol. At present, there are no solubility data or spectroscopic data for such inclusion complexes at constant pH in the literature. Therefore, we first investigated the effect of the natural CDs and then examined a series of modified CDs: sulfobutylether-β-cyclodextrin (SBE-β-CD), hydroxypropyl-α-cyclodextrin (HP-α-CD), hydroxypropyl-β-cyclo dextrin (HP-β-CD), hydroxypropyl-γ-cyclodextrin (HP-γ-CD) and methyl-β- cyclodextrin (Me-β-CD) on the solubility of Thp.
HP-α-CD, HP-β-CD, HP-γ-CD and Thp were purchased from Sigma-Aldrich (St. Louis, USA), α-CD, β-CD and γ-CD were purchased from Wako Chemical (Tokyo, Japan) and SBE-β-CD and Me-β-CD were purchased from Cydex (Kansas, USA) and the Junsei Chemical Co., Ltd. (Tokyo, Japan), respectively.
Phase solubility study
CD solutions of varying concentrations were made in a 10:90 (v/v) ethanol/50 mmol phosphate buffer mixture and were combined with excess amounts of Thp, after which the solutions were stirred at 300 rpm and 25°C. The concentration of Thp in each solution was subsequently measured using a fluorescence spectrometer following 3, 17, 24, 48 and 72 h. The data showed that an equilibrium concentration was obtained after 72 h, and so at that point the solutions were filtered through a 0.45 μm membrane. The concentration of Thp in each solution was determined based on fluorescence at λ = 287 nm with excitation at λ = 257 nm, using a Shimazu RF-5300PC spectrometer.
The pH of each sample solution was maintained at 7 by the addition of a 50 mmol/L potassium dihydrogen phosphate buffer. A 5.0 × 10-3 mol/L solution of Thp in ethanol was prepared and used in all experimental trials. In these trials, a 1 mL aliquot of this stock solution was transferred into a 10 mL volumetric flask together with an appropriate amount of a 1.0 × 10-2 mol/L and 1.25 × 10-2 mol/L CD phosphate solution, giving a final Thp concentration of 5.0 × 10-4 mol/L and CD concentrations of nil to 1.0 × 10-2 mol/L. Each solution was subsequently filtered through a 0.45 μm membrane. The fluorescence spectra of Thp (5.0 × 10-4 mol/L) was measured in a 10:90 (v/v) ethanol/50 mmol phosphate buffer solution. The fluorescence intensity generated by the Thp was found to vary depending on the concentration of CD added. Therefore, the value of K c could be obtained from the differences in the fluorescence intensities of the Thp/CD solutions. Fluorescence spectra were recorded with a Shimazu RF-5300 PC spectrometer.
Solid complexes of Thp and CDs were prepared by the following methods. Simple mixtures of Thp and CDs in solid form were prepared for comparison purposes.
Physical mixing method
Solid CD and Thp were combined at a ratio of 1 mmol (2.16 g in the case of the SBE-β-CD) to 1 mmol (0.3379 g for Thp) within a nylon bag, after which the mixture was shaken for 30 min.
Solid CD and Thp were combined at a ratio of 1 mmol (2.16 g for SBE-β-CD) to 1 mmol (0.3379 g for Thp) together with a small amount of ethanol (ca. 3 mL) and kneaded for 30 min.
Solid CD and Thp were combined at a ratio of 10 mmol (1.08 g for SBE-β-CD) to 10 mmol (0.168965 g for Thp) and dissolved in 50 mL of phosphate buffer (pH 7), following which the water was removed under vacuum at -40°C.
1H NMR measurements
A mixture of CD and Thp at a ratio of 10 mmol (0.0216 g for SBE-β-CD) to 10 mmol (0.0034 g for Thp) was dissolved in 1 mL of D2O to allow for 1H- and 1H-1H ROESY NMR measurements. When analyzing solely Thp, the compound was instead dissolved in CD3OD because of its poor solubility in water, using a ratio of 10 mmol of Thp to 1 mL of CD3OD.
1H-1H ROESY NMR data were obtained in the phase sensitive mode under continuous wave (CW) operation, with spin lock for mixing. Spectra of the inclusion complex were obtained by spin lock pulses of 180x-180-x with a steady-state sequence prior to d1, together with [grad]z-90x-[grad]z pulses.
X-ray diffraction (XRD)
Powder XRD patterns were obtained using a Rigaku Denki (Tokyo, Japan) Rint 2000 diffractometer with Ni-filtered Cu Kα radiation.
Differential scanning calorimetry (DSC)
The thermal behaviors of the solid complexes were assessed using a DSC 3100SA instrument (NETZSCH, Selb, Germany) at a heating rate of 10°C/min from 25 to 500°C.
Regression parameters and stability constants ( K ) for Thp/CD complexes as determined by solubility diagrams at pH 7
800 ± 50
680 ± 40
680 ± 80
1200 ± 200
The complexation ability of the SBE-β-CD appears to be the highest among the CDs investigated in this study.
where F and F 0 are the fluorescence intensities in the presence and absence of the CD, respectively, while [G], [CD] and a are the concentration of the guest compound (Thp), the concentration of the CD and a proportionality constant (Kondo et al. 1976; Hamai 1982), respectively.
The inset in Figure 3 demonstrates that a plot of 1/ΔF at 283 nm as a function of 1/[CD] for the β-CD data generates a straight line that in turn gives a K c value of 1200 L/mol.
Regression parameters and formation constants ( K c ) for Thp/CD complexes as determined from spectra acquired in a 10% (v/v) ethanol phosphate solution
6.4 × 10-3
1200 ± 40
5.4 × 10-3
700 ± 20
5.0 × 10-3
770 ± 40
1.9 × 10-3
2300 ± 200
The solubility of Thp in the presence of SBE-β-CD was increased by a factor of 11, and the stability of the resulting Thp/SBE-β-CD complex was higher than those of the other Thp/CD complexes. Because SBE-β-CD has sulfobutylether chains on both sides of CD rings, it has large hydrophobic space than the other derivatives of CDs, it would solubilize Thp. Furthermore, the end of sulfobutylether chain is anionic ion, which also increase the solubility of Thp/SBE-β-CD complex. Therefore, the structure of Thp/SBE-β-CD complex was investigated.
Structure of the inclusion complex
The above NMR data indicate that the Thp/SBE-β-CD complex consisted of an inclusion structure in the solution state. To further investigate this complex, a Thp/SBE-β-CD solid complex was subsequently synthesized and characterized.
Characterization of the Thp/SBE-β-CD solid complex
The solid systems obtained by physical mixing and kneading (Figures 7(c and d)) show six peaks at 2θ values of 6.07, 8.64, 12.1, 17.1, 17.9 and 24.0°, and five peaks at 2θ values of 5.85, 8.40, 11.9, 16.8 and 19.1° due to crystalline Thp, respectively. The solid obtained by the freeze-drying method, however, (Figure 7(e)) exhibits an amorphous state. These data suggest that the solid complex was obtained via freeze-drying and that Thp was included in the SBE-β-CD cavities.
In the case of the freeze-dried solid complex, the endothermic peak corresponding to the melting of Thp is not present, suggesting that the Thp interacts with the SBE-β-CD in the solid state to form an inclusion complex.
These results demonstrate that the solid complex obtained by the freeze-drying method was completely different from the systems generated using physical mixing and kneading. The Thp evidently interacts with SBE-β-CD in the solid complex obtained by the freeze-drying method, forming an inclusion complex.
The effects of natural and various modified CDs on the solubility of Thp were assessed, using the solubility method. The solubility of Thp in the presence of SBE-β-CD was found to be increased significantly, by a factor of approximately 11. The stoichiometry of each Thp/CD complex was observed to be 1:1. In the case of the Thp/SBE-β-CD inclusion complex, the formation constants (K c ) obtained by fluorometry was 2300 L/mol. The phenyl groups of the Thp were found to be included in the SBE-β-CD cavities. Finally, the freeze-drying method was determined to successfully generate solid inclusion complexes.
The authors thank Assistant Professor C. Tode of Kobe Pharmaceutical University for the measurements of 1H-1H COSY and 1H-1H ROESY NMR spectra.
- Ali SM, Shamim S (2014) Structure elucidation of benzhexol-β-cyclodextrin complex in aqueous medium by 1H NMR spectroscopic and computational methods. J Encapsul Adsorption Sci 4:63–70View ArticleGoogle Scholar
- Al-Rawashdeh NAF, Al-Sadeh KS, Al-Bitar MB (2010) Physicochemical study on microencapsulation of hydroxypropyl-β-cyclodextrin in dermal preparations. Drug Dev Ind Pharm 36(6):688–697View ArticleGoogle Scholar
- Al-Rawashdeh NAF, Al-Sadeh KS, Al-Bitar MB (2013) Inclusion complexes of sunscreen agents with β-cyclodextrin: spectroscopic and molecular modeling studies. J Spectrosc. doi:10.1155/2013/841409Google Scholar
- Alvarez-Parrilla E, De La Rosa LA, Torresrivas F, Rodrigo-Garcia J, González-Aguilar GA (2005) Complexation of apple antioxidants: chlorogenic acid, quercetin and rutin by β-cyclodextrin (β-CD). J Incl Phenom Maclocycl Chem 53:121–129View ArticleGoogle Scholar
- Benesi HA, Hildebrand JH (1949) A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. J Am Chem Soc 89:2703–2707View ArticleGoogle Scholar
- Haiyun D, Jianbin C, Guomei Z, Shaomin S, Jinhao P (2003) Preparation and spectral investigation on inclusion complex of β-cyclodextrin with rutin. Spectrochimica Acta Part A125:3421–3429View ArticleGoogle Scholar
- Hamai S (1982) Association of inclusion compounds of β-cyclodextrin in aqueous solution. Bull Chem Soc Jpn 55:2721–2729View ArticleGoogle Scholar
- Hibi T, Tatsumi M, Hanabusa M, Higuchi R, Imai T, Otagiri M, Uekama K (1984) Stabilization and reduction of irritant taste of anti-inflammatory drug pirprofen by β-cyclodextrin complexation. Yakugaku Zasshi 104(9):990–996Google Scholar
- Higuchi T, Connors KA (1965) Phase-solubility techniques. Adv Anal Chem Instrum 4:117–212Google Scholar
- Job P (1928) Formation and stability of inorganic complexes in solution. Ann Chem 9:113–203Google Scholar
- Kondo H, Nakatani H, Hiromi K (1976) Interaction of cyclodextrins with fluorescent probes and its application to kinetic studies of amylase. J Biochem 79:393–405Google Scholar
- Loftsson T, Duchêne D (2007) Cyclodextrins and their pharmaceutical applications. Int J Pharm 329:1–11View ArticleGoogle Scholar
- Maeda H, Onodera T, Nakayama H (2010) Inclusion complex of α-lipoic acid and modified cyclodextrins. J Incl Phenom Macrocycl Chem 68:201–206View ArticleGoogle Scholar
- Maeda H, Ogawa Y, Nakayama H (2013) Inclusion complex of melatonin and modified cyclodextrins. J Incl Phenom Macrocycl Chem 78:217–224View ArticleGoogle Scholar
- Mulisa E, Ndorbor T, Xiao D, He H (2014) Molecular docking method for the prediction of enantiorecognition of trihexyphenidyly and its derivatives on carboxy methyl-cylodextrins. Ind J Sci Res Tech 2(2):31–37Google Scholar
- Ono N, Miyamoto Y, Ishiguro T, Motoyama K, Hirayama F, Iohara D, Seo H, Tsuruta S, Arima H, Uekama K (2011) Reduction of bitterness of antihistaminic drugs by complexation with β-cyclodextrin. J Pharm Sci 100(5):1935–1943View ArticleGoogle Scholar
- ÖZdemir N, Ordu Ş (1998) Improvement of dissolution properties of furosemide by complexation with β-cyclodextrin. Drug Dev Ind Pharm 24:19–25View ArticleGoogle Scholar
- Reineccius TA, Reineccius GA, Peppard TL (2002) Encapsulation of flavors using cyclodextrins: comparison of flavor retention in alpha, beta, and gamma types. J Food Sci 67:3271–3279View ArticleGoogle Scholar
- Saenger W (1980) Cyclodextrin inclusion compounds in research and industry. Angew Chem Int Ed Engl 19:344–362View ArticleGoogle Scholar
- Szejtli J (1992) The properties and potential uses of cyclodextrin derivatives. J Inclusion Phenom Mol Recognit Chem 14:25–36View ArticleGoogle Scholar
- Uekama K (1985) Pharmaceutical applications of methylated cyclodextrins. Pharm Int 6:61–65Google Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.