the DEX-rich phase, respectively. Similarly, CPep, PEG and CPep, DEX represent the concentrations of Pep in the PEG-rich phase and the DEX-rich phase, ...
Solvent Extraction Research and Development, Japan, Vol. 20, 71 – 77 (2013)
Evaluation of the Hydrophilic and Hydrophobic Balance for the Complex between Indomethacin and Casein Hydrolysate Using an Aqueous Two-Phase System Tatsuya OSHIMA*, Asuka INADA and Yoshinari BABA Department of Applied Chemistry, University of Miyazaki, 1-1, Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan (Received December 29, 2012; Accepted February 8, 2013)
Complexation with casein hydrolysate (Pep) can be used for enhancing the water solubility of indomethacin. In the present study, the hydrophilic/hydrophobic balance of the complex (Indo-Pep) was evaluated using a poly(ethylene glycol)/dextran (PEG/DEX) aqueous two-phase system (ATPS). As the distribution ratio of Indo-Pep decreases with increasing Pep concentration, indomethacin was found to be more hydrophilic by complexation with Pep. The distribution ratio of Indo-Pep reached a minimum value when the pH was 7-8, where the distribution ratio of Pep showed a maximum value. From the results of the distribution ratios of Indo-Pep prepared using different components of Pep, the hydrophilic/hydrophobic balance of Pep seemed to influence the hydrophilic/hydrophobic balance of Indo-Pep.
1. Introduction Recently, a large number of drug candidates have arisen because of the high throughput screening of potential therapeutic agents [1, 2]. However, many of the drug candidates are poorly water-soluble and show low bioavailability when administered orally. In order to improve their aqueous solubility, various formulation techniques have been developed; complexation with macrocyclic cyclodextrins, surfactants, and hydrophilic polymers, as well as nano-emulsification [3-5]. A variety of formulation techniques for the poorly water soluble drug indomethacin (Figure 1) has been developed for the enhancement of its water-solubility. Complexation with hydrophilic or amphipathic additives is an effective technique for enhancing the solubility of indomethacin. Encapsulation of indomethacin into polymeric micelles has been developed [6, 7]. The formation of inclusion complexes with cyclodextrins is also available for
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enhancing the water solubility of indomethacin [8, 9]. Recently, complexation with a peptide mixture obtained as casein hydrolysate (Pep) was developed as a novel technique for the enhancement of the water-solubility of indomethacin [10]. The water-solubility of the complex (Indo-Pep) is much higher than that of indomethacin alone. From the results of the characterization of Indo-Pep, indomethacin was incorporated in the matrix of Pep. The size of Indo-Pep is quite small and it is able to pass through ultrafilter membranes. The water-soluble Indo-Pep should be more hydrophilic compared with indomethacin, although it is not easy to evaluate the hydrophilic/hydrophobic balance of the complex. In the present study, the hydrophilic/hydrophobic balance of Indo-Pep was evaluated from the distribution of Indo-Pep in an aqueous two-phase system (ATPS). Two immiscible phases of an ATPS are formed by combining two hydrophilic solutes (polymer/polymer or polymer/salt) in specific concentrations [11-13]. An ATPS has been used as a separation and purification medium for biomolecules such as proteins, due to the biocompatible hydrophilic environment. On the other hand, ATPSs can be applied for the characterization of the surface properties for biomolecules such as amino acids, peptides, protein, liposomes, and cells [14-16]. The mechanism of distribution in ATPSs is complex and not easy to predict, while the hydrophobicity of the target is one of the most important parameters [13]. The surface net hydrophobicity of protein (HFS) is directly related to the distribution ratio [14]. ATPSs can be used to estimate the hydrophobicity of the complex between small organic molecules and biomolecules [17, 18]. Therefore, the hydrophobicity of Indo-Pep was also evaluated using the poly(ethylene glycol)/dextran (PEG/DEX) ATPS. The effects of Pep quantity and pH on the distribution of Indo-Pep in the PEG/DEX ATPS were studied. Moreover, two components were separated from Pep and used to prepare Indo-Pep, in order to study the effect of the composition of Pep on the distribution of Indo-Pep in the ATPS.
2. Experimental 2.1 Materials Indomethacin, milk casein (Hammarsten grade), PEG-8,000 (PEG, average molecular weight = 8,000) dextran 40,000 (DEX, molecular weight = 32,000-45,000) were purchased from Wako Pure Chemical Industries, Ltd., (Osaka, Japan). Casein is a family of four phosphoproteins αS1-casein, αS2-casein, β-casein, and κ-casein [19]. In the present study, casein was used as the mixtures of phosphoproteins
Figure 1. Molecular structure of
as recei ved. α-C hy motr yps i n wa s pur c ha se d fr om
indomethacin.
Sigma-Aldrich(Tokyo, Japan). All other reagents are analytical grade. 2.2 Preparation of casein hydrolysate (Pep) Milk casein was enzymatically hydrolyzed using α-chymotrypsin at 45 °C for 20 h to obtain Pep. After lyophilization of the mixture, a white powder was obtained and used as Pep. Experimental details
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for the preparation of Pep are given in the literature [10]. From the results of gel filtration chromatography/HPLC analysis, Pep showed a wide molecular weight distribution with a range of roughly 1,000-10,000 [Da]. The average peptide length of Pep estimated by the 2,4,6-trinitrobenzene sulfonic acid (TNBS) method [20], was in the range of 4.0-5.2 because of the presence of smaller amino acids and peptides. 2.3 Preparation of Pep-S and Pep-P Pep (1.0 g) was dispersed in a mixture of acetone and water (2:1 v/v). The mixture was shaken (1000 rpm) using an EYELA cute Mixer CM-1000 (Tokyo, Japan) at room temperature for 5 min and cooled in an ice bath. After centrifugation of the cooled mixture at 6000 rpm for 15 min, the supernatant liquid and the precipitate was separated. The supernatant liquid and the precipitate were lyophilized to obtain Pep supernatant (Pep-S) and the Pep precipitate (Pep-P). 2.4 Preparation of Indo-Pep A 30 mM ethanol solution of Indo was prepared. Pep, Pep-S, or Pep-P (200 mg) was dissolved in distilled water and the pH was adjusted to 8.0 using 10 mM HCl and NaOH. The Indo solution and a Pep solution (5.0 cm3 each) were mixed and the mixture was shaken (1500 rpm), using a thermoshaker (Hangzhou Allsheng Instruments MSC-100, Zhejiang, China), at 30 °C for 4 h. After shaking, the mixture was lyophilized to obtain Indo–Pep, Indo-Pep-S, or Indo-Pep-P [10]. 2.5 Distribution test for Pep and Indo-Pep in the ATPS PEG (1.50 g), DEX (1.50 g), and distilled water (7.00 g) were mixed in a polypropylene centrifuge tube to form the ATPS. Pep, Pep-S, or Pep-P (10.0 mg) was added to the ATPS and the mixture was shaken at 1000 rpm at room temperature for 10 min. After shaking, the mixture was centrifuged at 6000 rpm for 5 minutes for phase separation. Both the upper phase (PEG-rich phase) and the lower phase (dextran-rich phase) were separated and their volumes were measured. The volumes of the upper phase and lower phase under typical conditions were 6.15 cm3 and 3.10 cm3, respectively. The concentrations of Pep originating from the tryptophan residues were analyzed using a UV-Vis spectrophotometer at 277 nm (JASCO V-660). In the similar manner, the distribution of Indo-Pep was examined by dissolving Indo-Pep, Indo-Pep-S, or Indo-Pep-P (5.0 mg) in the PEG-dextran ATPS (10.0 g). The pH of the aqueous solution was adjusted with small amounts of HCl and NaOH. The concentrations of Indo-Pep which originated from indomethacin were analyzed using a UV-Vis spectrophotometer at 320 nm. Distribution ratios for Indo-Pep (DIndo-Pep) and Pep (DPep) were calculated according to Eqs. (1) and (2). DIndo-Pep = CIndo-Pep, PEG/CIndo-Pep, DEX
(1)
DPep = CPep, PEG/CPep, DEX
(2)
where CIndo-Pep, PEG and CIndo-Pep, DEX represent the concentrations of Indo-Pep in the PEG-rich phase and the DEX-rich phase, respectively. Similarly, CPep, PEG and CPep, DEX represent the concentrations of Pep in the PEG-rich phase and the DEX-rich phase, respectively.
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3. Results and Discussion 3.1 Effect of the quantity of Pep on the water solubility and distribution in the ATPS for Indo-Pep In preliminary experiments, the distribution tests for Indo-Pep were carried out in the PEG/salt ATPS using NaH2PO4, MgSO4, (NH4)2SO4, and Na2SO4. However, Indo-Pep quantitatively distributed to the PEG-rich phase because of the higher hydrophobicity factor of the PEG/salt ATPS [14]. Therefore, distribution tests for Indo-Pep were carried out
4.5
using the PEG/DEX ATPS because of the lower 4.0
wavelength for Indo-Pep in the PEG-rich phase and the DEX-rich phase were the same (320 nm), the concentrations of Indo-Pep could be determined
KIndo-Pep [-]
hydrophobicity factor. As the maximum absorption
3.5 3.0
from the absorbance at 320 nm. 2.5
Figure 2 shows effect of the quantity of Pep on the distribution ratio of Indo-Pep in the PEG/DEX
2.0 0
ATPS. The distribution ratio of Indo-Pep decreases
complexation with a larger amount of Pep.
100
150
200
quantity of Pep [mg]
with increasing Pep concentration, suggesting that indomethacin becomes more hydrophilic through
50
Figure 2. Effect of the quantity of Pep on the distribution ratio of Indo-Pep in the PEG/DEX ATPS (PEG 15 wt%, DEX 15 wt%).
3.2 Effect of pH on the distribution of Pep and Indo-Pep The water solubility of Indo-Pep increases with increasing pH because of the deprotonation of the carboxylic acid group [10]. In order to study the factors for the complexation between indomethacin and Pep, the distribution of Pep and Indo-Pep as a function of pH was examined. The distribution tests were carried out in the pH range 6-8.5, because Indo-Pep is much less soluble under more acidic condition, and indomethacin would decompose under more basic conditions. Figure 3 shows effect of the pH on the distribution ratio of Pep. The distribution ratio of Pep reached a maximum value when the pH was 7-8. Pep should be more hydrophobic at the isoelectric point (pI). The pI of Pep, obtained as a casein hydrolysate, should be higher than that of casein (pI = 4.6) [19] because of the increase in the amount of amino groups and carboxyl groups by enzymatic hydrolysis. The distribution ratio of Pep might reach a maximum value at the pI although it is still not clear. Figure 4 shows the effect of the pH on the distribution ratio of Indo-Pep. The pH dependency of the distribution ratio of Indo-Pep is the inverse to that of Pep: The distribution ratio of Indo-Pep reached a minimum value when the pH was 7-8. The following hypotheses are suggested to explain the pH dependency: Indo-Pep should be more hydrophilic under higher pH conditions because of the deprotonation of the carboxylic acid groups. On the other hand, Pep is negatively charged and electrostatically repels the negatively charged Indo-Pep under basic conditions. Therefore, Indo-Pep can be excluded from the
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DEX-rich phase by Pep because the distribution ratio of Pep is less than 1 at pH values more than 8. 4.0
1.2
Indo-Pep
Pep
1.1
3.5 DIndo-Pep [-]
DPep [-]
1.0 0.9
3.0
0.8 2.5
0.7 0.6
2.0 5.0
6.0
7.0
8.0
9.0
5.0
6.0
7.0
8.0
9.0
pH (PEG-rich phase)
pH (PEG-rich phase)
Figure 3. Effect of the pH on the distribution ratio
Figure 4. Effect of the pH on the distribution ratio of
of Pep in the PEG/DEX ATPS (PEG 15 wt%, DEX
Indo-Pep in the PEG/DEX ATPS (PEG 15 wt%,
3
DEX 15 wt%): [Indo-Pep]=0.5 g/ cm3.
15 wt%): [Pep]=1.0 g/ cm .
3.3 Effect of the composition of Pep on the distribution of Pep and Indo-Pep Pep was separated into two components by dissolution in a mixture of acetone and water (2:1 v/v) as described in section 2.3. Pep-S which was soluble in the solution containing a large amount of acetone must be more hydrophobic than Pep-P. As shown in Table 1, the distribution ratio of Pep-S was higher than that of Pep-P due to higher hydrophobicity. Table 2 shows the distribution ratios of Indo-Pep-S and Indo-Pep-P which were prepared using Pep-S and Pep-P, respectively. The distribution ratio of Indo-Pep-S was higher than that of Indo-Pep-P. The results suggest that the hydrophilic/hydrophobic balance of Pep influences the hydrophilic/hydrophobic balance of Indo-Pep.
Table 1. Distribution ratios of Pep-S and Pep-P
Table 2. Distribution ratios of Indo-Pep-S and
in the PEG/DEX ATPS (PEG 12 wt%, DEX 15
Indo-Pep-P in the PEG/DEX ATPS (PEG 12 wt%,
wt%): [Pep]=1.0 g/ cm3.
DEX 15 wt%): [Indo-Pep]=0.5 g/ cm3.
Entry
DPep [-]
Entry
DIndo-Pep [-]
Pep-S
0.595
Indo-Pep-S
3.43
Pep-P
0.148
Indo-Pep-P
1.62
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4. Conclusions The hydrophilic/hydrophobic balance of Indo-Pep was evaluated using the PEG/DEX ATPS. The distribution ratio of Indo-Pep depended on the quantity of Pep and the pH. The results of the ATPS experiments suggest that indomethacin becomes more hydrophilic through complexation with Pep. However, a more detailed study should be conducted to understand the distribution mechanism of Indo-Pep. For past decades, complexation between poorly water soluble drugs and hydrophilic or amphipathic additives such as polymer micelles and macrocyclic compounds has been the developed to enhance the water solubility of the drugs. However, the hydrophilic/hydrophobic balance of the complexes had not been analyzed. ATPSs have the potential to provide an analytical method to evaluate the hydrophilic/hydrophobic balance of the complexes formed between poorly water soluble drugs and additives. Acknowledgment This research is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for Next Generation World-Leading Researchers (NEXT Program),” initiated by the Council for Science and Technology Policy (CSTP). A. I. was supported by the Sasakawa Scientific Research Grant from The Japan Science Society. References 1)
C. Leuner, J. Dressman, Eur. J. Pharm. Biopharm., 50, 47-60 (2000).
2)
C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, P.J., Adv. Drug Deliv. Rev., 46, 3-26 (2000).
3)
C. Leuner, J. Dressman, Eur. J. Pharm. Biopharm., 50, 47-60 (2000).
4)
R.G. Strickley, R.G. Pharm. Res., 21, 201-230 (2004).
5)
M.E. Brewster, T. Loftsson, Adv. Drug Deliv. Rev., 59, 645-666 (2007).
6)
M.-C. Jones, J.-C. Leroux, Eur. J. Pharm. Biopharm., 48, 101-111 (1999).
7)
V.P. Sant, D. Smith, J.-C. Leroux, J. Control. Release, 97, 301-312 (2004).
8)
B.W. Muller, U. Brauns, Int. J. Pharm., 26, 77-88 (1985).
9)
J. Szejtli, J. Incl. Phenom., 14, 25-36 (1992).
10) A. Inada, T. Oshima, H. Takahashi, Y. Baba, Int. J. Pharm., submitted. 11) M.R. Kula, Bioseparation, 1, 181-189 (1990). 12) R. Hatti-Kaul, Mol. Biotechnol., 19, 269-277 (2001). 13) J.A. Asenjo, B.A. Andrews, J. Chromatogr. A, 1218, 8826-8835 (2011). 14) R. Kuboi, H. Umakoshi, Solvent Extr. Res. Dev., Jpn., 13, 9-21 (2006). 15) K.X. Ngo, H. Umakoshi, T. Shimanouchi, R. Kuboi, Solvent Extr. Res. Dev., Jpn., 15, 133-137 (2008). 16) T. Shimanouchi, N. Shimauchi, K. Nishiyama, H.T. Vu, H. Yagi, Y. Goto, H. Umakoshi, R. Kuboi, Solvent Extr. Res. Dev., Jpn., 17, 121-128 (2010).
- 76 -
17) T. Oshima, A. Suetsugu, Y. Baba, Anal. Chim. Acta, 674, 211-219 (2010). 18) C. Kai, T. Oshima, Y. Baba, Solvent Extr. Res. Dev., Jpn., 17, 83-93 (2010). 19) S. Ahmad, F. Gaucheron, J.-F. Grongnet, A comparison of molecular changes in casein micelles: of buffalo and cow milk under different physico-chemical conditions. VDM Verlag Dr. Müller: Saarbrücken, Germany, (2010). 20) R. Fields, Methods Enzymol., 25, 464–468 (1972).
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