Controlling the mechanism of trypsin inhibition by the numbers of α-cyclodextrins and carboxyl groups in carboxyethylester-polyrotaxanes

Journal of Controlled Release 96 (2004) 301 – 307 www.elsevier.com/locate/jconrel

Controlling the mechanism of trypsin inhibition by the numbers of a-cyclodextrins and carboxyl groups in carboxyethylester-polyrotaxanes Masaru Eguchi, Tooru Ooya, Nobuhiko Yui * School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan Received 28 October 2003; accepted 21 February 2004

Abstract Carboxyethylester-polyrotaxanes (CEE-polyrotaxanes) with the various number of CEE-modified a-cyclodextrins (CEE-aCDs) were synthesized, and the effects of the number of CEE-a-CDs on calcium binding and trypsin inhibition were investigated. Calcium binding affinity was dependent on the density of the CEE groups accompanied with the number of a-CD threading in the CEE-polyrotaxanes. The high number of CEE-a-CDs leads to greater inhibition of trypsin activity than poly(acrylic acid), which is mainly due to the good calcium binding affinity. The CEE-polyrotaxane with the smallest number of CEE-a-CDs temporally interacted with trypsin, which was well correlated with the inhibition and recovery of trypsin activity. Therefore, the number of CEE-a-CDs in the CEE-polyrotaxanes can control the inhibition mechanism of trypsin activity. D 2004 Elsevier B.V. All rights reserved. Keywords: Polyrotaxanes; Calcium binding; Trypsin inhibition

1. Introduction Poly(acrylic acid) (PAA) based resins (Carbomer and polycarbophil) and methacrylate copolymers are well known absorption enhances for oral peptide delivery [1,2]. These resins have been shown to enhance the bioavailability of several peptides [3,4]. In addition to this function, PAA inhibits the activity of serine proteases such as trypsin and chymotrypsin [5– 10]. Some researches have suggested two kinds of the protease inhibitory mechanism. One is physicochemi-

* Corresponding author. Tel.: +81-761-51-1640; fax: +81-76151-1645. E-mail address: [email protected] (N. Yui). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.02.013

cal interactions such as electrostatic interaction and van-der Waals interaction [5]. This causes precipitation of trypsin, resulting in decreasing the trypsin concentration in the lumen. Another is denatulation of the enzymes by depletion of calcium from the enzyme structure [6,8,10]. The strength of calcium binding is dependent on the distance between carboxyl groups, which is strongly related to structural factors of polymeric backbones and cross-linked states. As a polymeric backbone of carboxyl groups, we have focused on polyrotaxanes, in which many a-cyclodextrins (aCDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with amino acid derivatives [11]. We previously synthesized a carboxyethylester-polyrotaxane (CEE-polyrotaxane) consisting of PEG, CEE-aCD and benzyloxycarbonyl (Z)-L-phenylalanine (Phe)

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and estimated the ability for trypsin inhibition [12]. Trypsin inhibition of the CEE-polyrotaxane was much greater than that of CEE-a-CD that is building block of the CEE-polyrotaxane. The carboxyl density in the CEE-polyrotaxane was significantly higher than the CEE-a-CD due to packing of CEE-a-CDs in the polyrotaxane structure. Therefore, the high density of CEE in relation to the polyrotaxane backbone seems to play an important role for the trypsin inhibition. However, the inhibition mechanism of the CEE-polyrotaxanes is still unclear. In this study, the number of CEE-a-CDs in the polyrotaxane structure varied to clarify the effect of the number of CEE-a-CDs on trypsin inhibition mechanism. Calcium binding affinity, trypsin activity and intermolecular complexation were discussed in terms of the number of CEE-a-CDs.

2. Materials and methods 2.1. Materials a-CD was purchased from Bio-Research of Yokohama (Yokohama, Japan). a-[(2-Amino-2-ethyl)-x-oxypripyl]-N-(amino-y-oxypropyl)polyethylene (x + y = 2.5) with a number average molecular ¯ n) of 2000 was kindly supplied from weight (M Suntechno Chemical (Tokyo, Japan) as JEFAMINE ED-2001 (PEG-BA2000). Poly(acrilic acid) (PAA, Mn 25,000), succinic anhydrite, N,N-diisopropylethylamine (DIEA) and calcium chloride were purchased from Wako (Osaka, Japan). Benzotiazol-1yloxytris(dimethylamino)phosohonium hexafluorophosphate (BOP regent), 1-hydroxybenzotriazole (HOBt), and 2-[N-morpholno]ethane-sulfonic acid (MES) were purchased from Nacalai Tesque (Kyoto, Japan). Poly(ethylene glycol) and benzyloxycarbonylL-tyrosine (Z-L-Tyr) were purchased from Kokusan Chemical (Tokyo, Japan). N-a-Benzonyl-L-arginine ethylester (BAEE) and N-a-benzonyl-L-arginine (BA) were purchased from Sigma (St. Louise, MO). Other chemicals used were of the highest purity available. 2.2. Synthesis of carboxyethylester-polyrotaxanes Synthetic route of the CEE-polyrotaxanes was shown in Scheme 1. A polypseudorotaxane consisting

Scheme 1.

of a-CDs and PEG-BA 2000 was prepared according to the method previously reported by Harada et al. [13]. PEG-BA 2000 (1 g, 5
10 4 mol) dissolved in water (5 ml) was added to a saturated aqueous solution of a-CD (11 g, 1.2
10 2 mol) at room temperature and ultrasonically agitated for 1 h, followed by stirred for 24 h. Capping reaction between the terminal amino-groups of the polypseudorotaxane and Z-L-Tyr was carried out using BOP reagent as a condensation reagent. The reaction itself has been reported by Tamura et al. [14]. Z-L-Tyr (3.9 g, 1.24
10 2 mol), BOP (5.5 g, 1.24
10 2 mol), HOBt (1.9 g, 1.24
10 2 mol) and DIEA (2.2 ml, 1.24
10 2 mol) were dissolved in DMF (10 ml). The mixture was introduced into a solution of the

M. Eguchi et al. / Journal of Controlled Release 96 (2004) 301–307

polypseudorotaxane (29 g, the number of a-CD: 22) in DMSO/DMF (see: Table 1, 20 ml). The mixture was stirred at room temperature for 6 h. The reaction mixture was poured into excess acetone to remove BOP, HOBt, DIEA and PEG-BA. The precipitate was collected by centrifugation and washed with ethanol and distilled water to remove impurities. The resulting precipitate was collected by centrifugation and dried in vacuo at room temperature to obtain a Z-L-Tyrterminated polyrotaxane as white powder. Table 1 summarizes the synthetic results. 1 H-NMR (D2O + NaOD, ppm): d 7.35– 7.18 (aromatics of Z-LTyr), 4.76 (C(1)H of a-CD), 3.84 – 3.23 (C(3), C(5), C(6)H, C(4)H and C(2)H of CD), 3.50 (CH2 of PEG). Carboxyethylester-polyrotaxanes (CEE-polyrotaxanes) were prepared according to our method [12]. Briefly, the Z-L-Tyr-terminated polyrotaxanes and succuinic anhydride (same mol of hydroxyl groups in the Z-L-Tyr-terminated polyrotaxanes) were dissolved in dry pyridine and stirred at room temperature. The reaction mixture was poured into excess ether and washed with ether three times. The precipitate was collected by centrifuging and dried under in vacuo to give the CEE-polyrotaxanes. The degree of substitution of CEE groups in the polyrotaxane was estimated from the ratio of the methylene peak of CEE (2.28 ppm) and C(1)H of a-CD (4.88 ppm) on the 1HNMR spectra. 1H-NMR (D 2O + NaOD, ppm): d 7.35 –7.18 (aromatics of Z-L-Tyr), 4.88 (C(1)H of aCD), 4.00 –3.30 (C(3), C(5), C(6)H, C(4)H and C(2)H of CD), 3.49 (CH2 of PEG), 2.50 – 2.00 (CH2 of CEE). 2.3. Calcium binding assay The CEE-polyrotaxanes (0 – 5.5
10 2 M) were dissolved in 50 mM MES aqueous solution adjusted to pH 6.7 with 1 M KOH (MES/KOH buffer, pH 6.7) containing calcium chloride (100 mg/ml), and stirred Table 1 Preparation of Z-L-Tyr terminated polyrotaxanes Sample code

Solvent No. of % of Total (DMF/ a-CDa a-CD Mna DMSO) (calculated) threading

22a/E2-Tyr-Z 100/0 18a/E2-Tyr-Z 90/10 15a/E2-Tyr-Z 85/15 a

22 (22) 18 (22) 15 (22)

100 82 68

Calculated from the 1H-NMR spectra.

Yield (%)

24,000 30 20,100 15 17,200 5.5

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for 2 h at room temperature. The concentration of unbound calcium ([Ca2 +]free) was determined using a Ca2 +-sensitive electrode (HORIBA, Japan). The concentration of bound calcium ([Ca2 +]bind) was calculated by the following equation: ½Ca2þ bind ¼ ½Ca2þ total  ½Ca2þ free where [Ca2 +]total is the total concentration of Ca2 +. 2.4. Trypsin inhibition study Trypsin inhibition was estimated according to the previous study by Lueßen et al. [6]. The amount of 1.5 mmol N-a-benzoyl-L-arginine (BAEE) was dissolved in MES/KOH buffer (pH 6.7) including the CEEpolyrotaxanes (20 ml). Five milliliter of the different substrate dilutions was used for one degradation experiment. The degradation was started by adding 15900 IU trypsin/ml stock solution to the mixtures at 37 jC (final trypsin concentration: 24 IU/ml). In order to analyze the degradation using high performance liquid chromatography (HPLC), the reaction mixture (50 Al) was sampled at an appropriate time and diluted in 1 ml phosphoric acid (pH 2.0) to stop trypsin activity. The degradation product (N-a-benzoyl-L-arginine, BA) was determined by the HPLC with a reversed-phase column (COSMOSIL 5C18-AR-II, 250
4.5 mm2; Nacalai Tesque, Kyoto Japan) at a follow rate 0.75 ml/min. The mobile phase consists of: elute A, 86% (v/v) 10 mM ammonium acetate (pH 4.2) and 14% (v/v) acetonitrile and elute B, 20% (v/v) 10 mM ammonium acetate (pH 4.2) and 80% (v/v) acetonitrile. Gradient elution was performed as follows: 0 – 8 min: 92% A/8% B, isocratic; 8– 10 min: 50% A/50% B, linear gradient; 10 – 13 min: 50% A/ 50% B, isocratic. BA was detected at 253 nm. In this condition, the elution peak of BA was found to be 6.35 min. 2.5. Evaluation of physical interactions between trypsin and carboxyethylester-polyrotaxanes The MES buffer containing CEE-polyrotaxanes (concentration of carboxyl groups was 25 mM, 2 ml) was prepared in an UV –Vis cuvette. Stock solution of trypsin (15900 IU/ml) was added to be 24 IU/ml as final concentration, and immediately started to mea-

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sure transmittance at 500 nm by using an UV – Vis spectrometer (V-550, Jasco, Tokyo, Japan).

3. Results and discussion 3.1. Preparation of carboxyethylester-polyrotaxanes having various number of a-CDS Z-L-Tyr was selected as the terminal moiety to prevent a-CD dethreading since the phenol side-group of L-Tyr is larger than a-CD cavity. BOP reagent was used for coupling reagent between the terminal amino group of PEG-BA and carboxyl group of Z-L-Tyr, since the condensation reaction using the BOP reagent is faster than those condensation reactions of succinimide ester [15]. The synthetic conditions and the results of CEE-polyrotaxanes were summarized in Table 1. The number of a-CDs per polyrotaxane molecule was varied from 15 to 22, which was controlled by changing the mixing ratio of DMF and DMSO at the capping reaction. It is known that DMSO eliminates hydrogen bonding between aCDs in polypseudorotaxanes consisting of a-CDs and PEG [13]. With increasing the mixing ratio of DMSO and DMF, the number of a-CDs decreased as expected. The stoichiometric number of a-CD threading on to the PEGs of Mn 2000 is 22. It is considered that a-CD dethreading from the PEG chain is controlled by eliminating rate of hydrogen bonding in polyseudorotaxane. The obtained polyrotaxanes and succinic anhydride were allowed to react for 24 h to obtain CEE-polyrotaxanes. It was found that the number of CEE-aCDs per CEE-polyrotaxane molecule was varied 11, 16 and 22 when the number of a-CDs in the used polyrotaxanes was 15, 18 and 22, respectively (Table 2). This indicates that small amount of a-CD in the polyrotaxane was dethreaded during the reaction. In this condition, six CEE groups were introduced to one a-CD molecule in all the CEE-polyrotaxanes. 3.2. Effect of the polyrotaxane structure, a-CD threading percentage and CEE numbers on calcium ion binding Calcium is an important ion for maintaining the thermodynamic stability for trypsin [16,17].

Table 2 Synthesis of carboxyethylester-polyrotaxanes Sample codea

No. of CEE/molb

No. of a-CD/molb

% of a-CD threading

Total Mnb

132CEE-22a/ E2-TYR-Z c 96CEE-16a/ E2-TYR-Z c 66CEE-11a/ E2-TYR-Z c

132

22

100

37,200

96

16

72

27,800

66

11

50

19,900

a CEE-a/E-TYZ-Z: Z-L-Tyr terminated carboxyethylester polyrotaxane. E2: PEG-BA2000. b Calculated from the 1H-NMR spectra. c 132CEE-22a/E2-TYR-Z, 96CEE-16a/E2-TYR-Z and 66CEE11a/E2-TYR-Z were synthesized from 22a/E2-TYR-Z, 18a/E2TYR-Z and 15a/E2-TYR-Z, respectively.

Poly(acrylic acid) derivatives, Carbomer and polycarbophil have been shown to inhibit serine proteases such as trypsin due to calcium binding at pH 6.7 [6,10]. It is believed that calcium binding affinity of the PAA derivatives is dependent on the distance of carboxyl groups, which is changeable by grating PAA chain on some polymeric backbones and cross-link density of the PAA derivatives [10]. The enzymatic inhibition is directly related to the calcium binding affinity of the inhibitors [6]. Fig. 1 shows the bound calcium to 132CEE-22a/ E2-TYR-Z, 96CEE-16a/E2-TYR-Z, 66CEE-11a/E2TYR-Z and PAA as a function of concentration of carboxyl groups per total calcium concentration ([COOH]/[Ca2 +]total). The 132CEE-22a/E2-TYR-Z bound ca. 88% calcium when [CEE]/[Ca2 +]total was 13, although the 96CEE-16a/E2-TYR-Z and 66CEE11a/E2-TYR-Z bound ca. 58% calcium at the same [CEE]/[Ca2 +]total. PAA bound ca. 95 % calcium when [CEE]/[Ca2 +]total was 13. Calcium binding affinity was in the following order: PAA>132CEE22a/E2-TYR-ZH96CEE-16a/E2-TYT-Z>96CEE11a/E2-TYT-Z. These results suggest that the number of CEE-a-CD can modulate the calcium binding affinity. 3.3. Effect of CEE-polyrotaxanes on inhibitory of trypsin activity In order to estimate the effect of trypsin inhibition activity, trypsin-catalyzed degradations of BAEE in the presence of those compounds were examined.

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Fig. 1. Calcium binding affinity of o: 132CEE-22a/E2-TYR-Z, 5: 96CEE-16a/E2-TYR-Z, 5: 66CEE-11a/E2-TYR-Z trypsin, and n: PAA (mean F S.E.M., n = 3).

Fig. 2 shows release profiles of BA (degradation product of BAEE) as a function of time. Here, the concentration of carboxyl groups was unified. The amount of BA release was proportional to time until 30 min in any case of the CEE-polyrotaxanes as well as the case of PAA. As seen in the initial slope of BA release curves, the initial rate of BA release in the presence of 132CEE-22a/E2-TYR-Z was much slower than the other CEE-polyrotaxanes. With decreasing the number of CEE-a-CDs (99CEE-16a/ E2-TYR-Z) the initial rate increased, but the CEEpolyrotaxane with the smallest number of CEE-aCDs (66CEE-22a/E2-TYR-Z) showed almost similar kinetics to 132CEE-22a/E2-TYR-Z. Generally, in the case of PAA-based resins, trypsin activity is inhibited by depletion of calcium out of the trypsin structure at the neutral pH region due to ionization of carboxyl groups. On the other hand, decreasing pH around four leads to protonation of the carboxyl groups, resulting in enhanced trypsin-polymer interactions rather than calcium binding [6]. From this fact, the inhibitory effect of PAA, seen in Fig. 2, should be mainly due to depletion of calcium out of the trypsin structure. If the linear curves of the initial BA release in Fig. 2 are hypothesized to indicate the calcium depletion, the initial rate might be correlated with calcium binding affinity. However, the rate was

305

not proportional to the calcium binding affinity (Fig. 1): the CEE-polyrotaxane with less calcium binding affinity (66CEE-11a/E2-TYR-Z) showed similar trypsin inhibition to that with high calcium binding affinity (132CEE-22a/E2-TYR-Z). These results suggest that the strong inhibition of trypsin activity at the initial stage by the CEE-polyrotaxane with small number of CEE-a-CDs (66CEE-11a/E2-TYR-Z) is due to not only the calcium depletion but also another factor. Interestingly, the amount of BA in the solution containing 66CEE-11a/E2-TYR-Z drastically increased after 30 min (Fig. 2). This means that trypsin activity is recovered by some reasons. One of possible reasons of the spontaneous activity change might be changing extent of trypsin-polymer interaction that is considered as another factor of trypsin inhibition. In order to assess the extent of trypsin-polymer interactions, viscosity of the all the solutions in the same experimental conditions was measured. There were no differences in the viscosities between the solutions (data not shown), indicating that no physical crossliking occurs in trypsin-polymer interactions. It is noted that only the solution of 66CEE-11a/E2-

Fig. 2. Release profiles of BA (degradation product of BAEE) in the presence of trypsin (mean F S.E.M., n = 3). 5: control (only BAEE), o: 132CEE-22a/E2-TYR-Z, 5: 96CEE-16a/E2-TYR-Z, 5: 66CEE-11a/E2-TYR-Z and n: PAA25. Concentrations of COOH groups in each solution were 25 mM. Trypsin concentration was 24 IU/ml.

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4. Conclusion

Fig. 3. Transmittance change of solution containing CEE-polyrotaxanes and PAA25 (COOH: 25 mM) after trypsin addition (24 IU/ml). o: 132CEE-22a/E2-TYR-Z, 5: 96CEE-16a/E2-TYR-Z, 5: 66CEE-11a/E2-TYR-Z and n: PAA25.

The CEE-polyrotaxanes with different number of CEE-a-CDs were synthesized and these inhibitory effects on trypsin activity were evaluated. Calcium binding affinity increased with the number of CEEa-CDs, indicating that density of carboxyl groups along with the polyrotaxane backbone is controllable factor of calcium binding affinity. The CEE-polyrotaxanes with the smallest number of CEE-a-CDs showed temporal interaction with trypsin, which might be main inhibition mechanism. The CEEpolyrotaxanes with the highest number of CEE-aCDs showed greater inhibition of trypsin activity than PAA. Therefore, the mechanism and extent of trypsin inhibition can be controllable by the design of CEE-polyrotaxanes.

Acknowledgements TYR-Z seemed to become turbid, so that transmittance of the solution was then measured (Fig. 3). As shown in Fig. 3, only the solution of 66CEE-11a/E2TYR-Z slightly decreased the transmittance 10 min after mixing, and then increased again. Since the transmittance change of the other CEE-polyrotaxanes and PAA was not observed, the small number of CEEa-CDs contributed to increasing the extent of trypsinCEE-polyrotaxane interactions. Taking the spontaneous recovery of trypsin activity in the presence of 66CEE-11a/E2-TYR-Z (Fig. 2) into account, the temporal interaction between trypsin and 66CEE11a/E2-TYR-Z is likely to be the main mechanism of trypsin inhibition. Walker et al. reported that carbomer inhibits trypsin activity due to non-specific interactions between trypsin and carbomer at pH7.0 [5]. On the other hand, Luehen et al. reported that carbomer and polycarbophil could hardly inhibit trypsin activity due to the non-specific interactions at the neutral pH [10]. In the case of CEE-polyrotaxanes, the extent and the way of the inhibition could be changeable by the number of CEE-a-CDs. In addition, the inhibitory effect of CEE-polyrotaxane with the highest number of CEE-a-CDs was greater than that of PAA (Fig. 2). Presumably, not only the depletion of calcium out of trypsin structure but also denaturation of trypsin by physical interactions contributes to the inhibitory effect.

This study was financially supported by a Grant-inAid for Scientific Research (B) (No. 14380397), from the Ministry of Education, Science, Sports, and Culture, Japan, and by the 21st century COE program ‘‘Scientific Knowledge Creation Based on Knowledge Science’’, Japan Advanced Institute of Science and Technology.

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