Chitosan−Pentaglycine−Phenylboronic Acid Conjugate - American

Bioconjugate Chem. 2006, 17, 1000−1007

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Chitosan-Pentaglycine-Phenylboronic Acid Conjugate: A Potential Colon-Specific Platform for Calcitonin Reem Smoum,†,§ Abraham Rubinstein,*,‡ and Morris Srebnik*,† Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, and Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel. Received December 19, 2005; Revised Manuscript Received June 7, 2006

Novel drug delivery vehicles based on the biodegradable, mucoadhesive polysaccharide chitosan covalently linked to a boronic acid protease inhibitor have been prepared and characterized. It was anticipated that these conjugates could protect a proteinaceous drug, such as salmon calcitonin, against proteolysis by serine proteases, an obstacle that prevents its oral administration. Specifically, 4-formylphenylboronic acid was linked to chitosan. Three types of conjugates were prepared. In the first, 4-formylphenylboronic acid was directly linked to chitosan. The other two conjugates employed glycylglycine and pentaglycine spacers. Enzyme-inhibition assays toward trypsin and elastase, in the presence of the enzyme chitosanase, demonstrated a strong inhibitory effect for the chitosanpentaglycine-phenylboronic acid conjugates, while no inhibitory effect could be detected without chitosanase. The chitosan-pentaglycine-phenylboronic acid conjugate with the highest degree of substitution of 4-formylphenylboronic acid was able to decrease the salmon calcitonin degradation rate by trypsin. It is concluded that chitosan-pentaglycine-phenylboronic acid conjugates are a potential multifunctional, colon-specific vehicle for orally administered salmon calcitonin.

INTRODUCTION Oral administration of peptide and protein drugs is a challenge because of their large size, their susceptibility to luminal and brush border proteolysis, and their limited stability (1). Consequently, protein drugs exhibit poor oral bioavailability (2). To increase their availability into the body after oral administration, proteinaceous drugs require absorption enhancers, protease inhibitors, or both (3). In this context, the colon is an interesting organ in the gastrointestinal (GI) tract. In addition to its improved ability to respond to absorption enhancers (4), reports in the literature indicate that proteolysis occurs into a lesser extent in the human colon compared with the small intestine (5). It was, therefore, suggested that colon-specific drug carriers could facilitate the oral absorption of proteinaceous drugs. Still, even the relatively lower proteolytic activity of the colon requires that protein drugs should be protected due to the longer residence time in the this organ (6). If protection is successful, the prolonged residence time could turn out to be advantageous because the drug would be exposed for extended periods of time to the absorptive epithelium. An efficient drug carrier would be the one that would provide ongoing protection over time. One possible solution is the development of synchronous release polymeric drug platforms, which would release drugs and enzyme inhibitors in a concomitant manner (7). An attractive alternative to the synchronous release formulations is the one in which the protease inhibitor is covalently bound to the drug carrier. In a recent study, antipain, chymostatin, elastatinal, and Bowman-Birk inhibitors were each covalently attached, together with ethylenediamine tetraacetic acid (EDTA), to chitosan * To whom correspondence may be addressed. Morris Srebnik: mailng address POB 12065, 91120, Jerusalem, Israel; fax +972-2-6758201; tel +972-2-675-7301; e-mail [email protected]. Abraham Rubinstein: mailing address POB 12065, 91120, Jerusalem, Israel; fax +972-2-675-8201; tel +972-2-675-7301; e-mail [email protected]. † Department of Medicinal Chemistry and Natural Products. § E-mail: [email protected]. ‡ Department of Pharmaceutics.

to guarantee the inactivation of the pancreatic serine proteases, membrane-bound Zn-dependent peptidases, and carboxypeptidase A and B (8). Boronic acids form reversible covalent adducts with the active site of serine proteases. The tetrahedral geometry of boron coordination mimics the high-energy tetrahedral geometry on the reaction center carbon during the hydrolysis reaction (9). For this reason, boronic acid inhibitors are often considered to be transition state analogue inhibitors. Peptide boronic acid inhibitors are usually tri- or tetrapeptides with a boronic acid group in place of the C-terminal carboxylate (10). When the peptide composition is based on the substrate specificity of a particular protease, the inhibitor combines the favorable geometry of the adduct formed by the boron atom in the active site with the enzyme-substrate interactions of the peptide chain. In this context, we propose to design new drug delivery vehicles based on a biodegradable polysaccharide linked to a peptide boronic acid. It is anticipated that such compounds could avoid protein hydrolysis by serine proteases such as trypsin and chymotrypsin. A variety of vehicles have been developed to allow colonspecific delivery of drugs (11). Special efforts have been dedicated in our group to fermentable polysaccharides (12). Chitosan, a natural polysaccharide, is made of N-acetyl-Dglucoamine and D-glucosamine and the N-acetyl-2-amino-2deoxy-D-glucopyranose units linked by (1 f 4)-β-glycosidic bonds. In the past decade, it became popular as a drug delivery vehicle due to its biocompatibility, degradation properties, and ability to be conjugated with a variety of substrates via its amine groups (13). Tozaki and co-workers have identified its ability to degrade in the human colon and suggested it as a colonspecific vehicle (14). We, therefore, hypothesized that if a protease inhibitor was conjugated to chitosan, a novel colon-specific platform could be produced for protein drugs. In this study, 4-formylphenylboronic acid was found to inhibit the R-chymotrypsin, trypsin, elastase, and leucine aminopeptidase, and thus was selected as

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Chitosan−Pentaglycine−Phenylboronic Acid Conjugate

the protease inhibitor that could be covalently attached to chitosan. The protein drug chosen was salmon calcitonin (sCT). Calcitonin is a 32 amino acid polypeptide used in the treatment of postmenopausal osteoporosis and Paget’s disease and in the management of hypercalcemia (15). The current mode of administration of commercial sCT is intranasally, a route that suffers from large variability in bioavailability (0.3-30%) (16). For this reason, it is appealing to administer it orally if the barrier of serine proteases is overcome. The specific goals of the present study were to (a) conjugate phenylboronic acid to chitosan with and without spacers, (b) study, in vitro, the inhibition properties of the polysaccharide conjugates, and (c) assess the possible protective effect of the optimal polysaccharide inhibitor conjugate(s) toward a model protein drug, sCT.

EXPERIMENTAL PROCEDURES Chitosan [DS ) 85% glucosamine (GlcN), 15% N-acetylglucosamine (GlcNAc)], trypsin, R-chymotrypsin, elastase, N-benzoyl-arginine-p-nitroanilide, N-benzoyl-tyrosine-p-nitroanilide, N-succinyl-(Ala)3-p-nitroanilide, L-leucine-p-nitroanilide, 1-ethyl-3,3-dimethylaminopropyl carbodimide (EDAC), and sodium borohydride (NaBH4) were all purchased from Sigma-Aldrich. Salmon calcitonin (sCT) was obtained from CALBIOCHEM. Solvents from Biolab were purchased as analytical grade. HPLC-grade trifluoroacetic acid (TFA) and acetonitrile were obtained from Biolab. All solutions and buffers were prepared with deionized water. All other materials were of reagent grade. 1H, 13C, and 11B NMR was recorded on a Varian 300 MHz instrument at frequencies of 300, 75.9, and 96.29 MHz, respectively. Shift values were reported with respect to TMS (1H, 13C) and BF3‚OEt2 (11B). Infrared spectra were run for samples as KBr disks on a Bruker Vector 22 FT-IR spectrophotometer. Biological assays were performed on a UVIKONxs-BIO-TEK instrument. Synthesis and Purification of Chitosan-Phenylboronic Acid Conjugates. Chitosan (50 mg, 0.3 mmol of NH2) was dissolved in 5 mL of 1% acetic acid, and the mixture was stirred for 1 h to obtain a 1% (m/v) solution. Various amounts of 4-formylphenylboronic acid (Table 1) dissolved in 3 mL of methanol were added to the solution, which was stirred at room temperature. After 1 h, sodium borohydride (NaBH4) (1.5%) in 2 mL of methanol was added. The reaction mixture was then incubated for 24 h at room temperature under continuous stirring. The precipitate formed was filtered and washed with water and methanol thoroughly. Selected data for 3 are as follows: 1H NMR (1% CD3COOD in D2O) δ 1.899 (s, NHAc), 3.012 (br m, H-2 of GlcN), 3.5913.748 (br m, H-2,3,4,5,6 of β-D-glucose (Glc)), 4.577 (br, -NHCH2Ph), 4.726 (br, H-1 of β-D-Glc), 7.36 (br, H-2,6 of Ar), 7.705 (br, H-3,5 of Ar). FT-IR (KBr, cm-1) 3359, 2876, 1654.95, 1610.48, 1560.75, 1411.74, 1376.74, 1261.59, 1070.44, 65.17. N-Succinylation of the Chitosan-Phenylboronic Acid Conjugates. Chitosan-boronic acid conjugate (20 mg) was dissolved in 2 mL of 1% acetic acid, and then the solution was diluted with 2 mL of methanol. Succinic anhydride (6 equiv ) 72 mg) was added to the diluted solution and stirred at room temperature (rt). After the mixture was stirred for 24 h, the pH of the mixture was adjusted to 5.0 with 5% w/v aq NaOH to give a precipitate. The precipitate was collected by filtration and dispersed in water. The pH of the dispersion was adjusted to 10-12 with 5% w/v aq NaOH to give a solution. The solution was dialyzed using dialysis membrane (molecular weight cut off, 12 000-14 000) at room temperature for 2-3 days and lyophilized.

Table 1. Preparation of Conjugates 3 with Aldehyde 2 entry

2 (equiv)

DSa

recoveryb (wt %, g/g)

solubility in H2O

1 2 3 4 5

0.4 0.8 1.0 2.0 3.0

46.9 51.2 53.0 56.0 58.0

46.9 51.9 53.0 78.2 79.9

no no no no moderate

elemental analysis C H N 42.03 42.19 44.11 45.34 46.02

6.97 6.91 6.90 6.71 6.60

6.12 5.61 5.41 5.14 4.94

a DS (degree of substitution) was determined from the peak area of phenyl protons (δ 7.0-7.4) and H-2 of GlcN and N-alkylated GlcN residues (δ 3.2) and from elemental analysis. b Recovery (%) was obtained by dividing the obtained product weight by the weight of the chitosan: Recovery (%) ) [wt of product (g)/wt of DAC 85%] × 100.

N-Succinyl-conjugated chitosan (DS ) 32.5%). 1H NMR (D2O) δ 2.085 (s, -NH(CO)CH2), 2.3 (br s, -NH(CO)CH2 and -CH2COONa), 3.602 (br m, H-2,3,4,5,6 of GlcN), 7.259 (br, H2,6 of Ar), 7.620 (br, H3,5) of Ar. Synthesis of Glycylglycine-Phenylboronic Acid Conjugates. To a 10 mL solution of glycylglycine (2.645 g, 20 mmol) in water containing NaOH (0.405 g, 10 mmol), ethanol (10 mL) was added. Then, 4-formylphenylboronic acid (1.5 g, 10 mmol) dissolved in 2 mL of ethanol was added. The resulting solution was stirred at room temperature for 30 min and then cooled to ca. 2 °C. Sodium borohydride (0.460 g, 12 mmol) in ethanol (5 mL), containing a few drops of a 2 M NaOH solution was then slowly added, a colorless solution being obtained. A few drops of 3 M HCl were added till pH 4-5 was reached, and the mixture was left overnight with stirring. A white precipitate formed, was collected by filtration, and was washed with cold water and diethyl ether. To this white solid, water at ca. 40 °C was added; the mixture was stirred for a few minutes and left at ca. 4 °C for several hours. The solid was filtered, washed with water and diethyl ether, and dried. Yield: 52.60%. 1H NMR (CD3OD): δ 3.723 (2H, s, CH2COOH), 3.796 (2H, s, CH2NH), 4.172 (2H, s, CH2-phenol), 7.448 (2H, d, CH aromatic), 7.758 (2H, d, CH aromatic). 13C NMR (CD3OD) 42.775, 51.110, 128.761, 132.240, 133.642, 134.341, 166.028, 174.462. 11B NMR 30.791. Anal. (C11H15BN2O5) C, H, N. Synthesis of Pentaglycine-Phenylboronic Acid Conjugates. The procedure was similar to that of glycylglycinephenylboronic acid. Yield: 64.46%. 1H NMR (DMSO): δ 3.099 (2H, s, CH2COOH), 3.674 (2H, s, CH2NH), 3.713 (2H, s, CH2NH), 3.750 (2H, s, CH2NH), 3.768 (2H, s, CH2NH), 4.439 (2H, s, CH2-phenol), 7.284 (2H, d, CH aromatic), 7.707 (2H, d, CH aromatic). 13C NMR (DMSO) 41.894, 42.509, 42.737, 45.010, 51.717, 53.144, 127.892, 134.746, 142.037, 166.795, 169.501, 169.790, 169.968, 171.733, 172.044. 11B NMR 30.791. Anal. (C17H24BN5O8) C, H, N. Synthesis and Purification of Chitosan-Spacer-Phenylboronic Acid Conjugates (Spacer ) Glycylglycine or Pentaglycine). The covalent attachment of the spacer-phenylboronic acid conjugates to chitosan was achieved by the formation of amide bonds between amino groups of the polymer and the terminally located carboxylic acid group of the spacerphenylboronic acid inhibitor. The general procedure was as follows: Chitosan (50 mg, 0.3 mmol NH2) was dissolved in 5 mL of 1% acetic acid solution, and the mixture was stirred for 1 h to obtain a 1% (m/v) solution. Increasing amounts of spacer-phenylboronic acid conjugate (glycylglycine-phenylboronic acid in 3 mL of methanol or pentaglycine-phenylboronic acid in 2 mL of H2O) were added to the solution, which was stirred at room temperature. After 1 h, 1-ethyl-3,3dimethylaminopropyl carbodimide (EDAC) (1:1 mole ratio of EDAC/glycylglycine-phenylboronic acid; 2:1 mole ratio of EDAC/pentaglycine-phenylboronic acid) in 2 mL of methanol was added to mediate the formation of amide bonds between the amino groups of chitosan and the carboxyl groups of the

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spacer-phenylboronic acid conjugate. The reaction mixture was then incubated for 24 h at room temperature under continuous stirring. The resulting polymer conjugate was isolated by exhaustive dialysis against deionized water for 2 days. Then the methanol was evaporated, and the purified product was lyophilized. The yellow solid (film) obtained was stored at room temperature until use. 1H NMR (D2O) for chitosan-glycylglycine-phenylboronic acid conjugates δ 2.924 (H, br m, H2 of GlcN), 3.587 (10H, br m, H-2,3,4,5,6 of β-D-glucose (Glc), CH2COOH and CH2NH), 4.172 (2H, s, CH2-phenol), 7.104 (2H, br m, CH-aromatic), 7.546 (2H, br m, CH-aromatic). FT-IR (KBr, cm-1) 3421.15, 2926.78, 1653.77, 1637.15, 1560.03, 1409.70, 1377.45, 1316.99, 1259.65, 1154.28, 1073.13, 898.09, 653.53). Synthesis and Purification of Chitosan-PentaglycinePhenylboronic Acid Conjugates. The procedure for the synthesis and purification of chitosan-pentaglycine-phenylboronic acid conjugates is similar to that of the chitosanspacer-phenylboronic acid conjugates. 1H NMR (D2O) for chitosan-pentaglycine-phenylboronic acid conjugates δ 1.877 (s, NHAc), 2.669 (H, br m, H2 of GlcN), 3.324 (6H, br m, H-2,3,4,5,6 of β-D-glucose (Glc)), 3.481 (H, s, CH2CONH), 3.594 (2H, s, CH2NH), 3.953 (2H, s, CH2NH), 4.058 (2H, s, CH2-phenol), 7.163 (2H, br s, CH-aromatic), 7.595 (2H, br s, CH-aromatic). Determination of the Degree of Modification. 1H NMR spectroscopy and elemental analysis were used to quantify the amount of phenylboronic acids, glycylglycine-phenylboronic acids and pentaglycine-phenylboronic acids conjugated to chitosan. In Vitro Evaluation of the Protective Effect of 4-Formylphenylboronic Acid, Glycylglycine-Phenylboronic Acid, and Pentaglycine-Phenylboronic Acid Conjugates toward Serine Proteases and Leucine Aminopeptidase. R-Chymotrypsin, type II (EC 3.4.21.1) from bovine pancreas, was purchased from Sigma (MW 25 000). The inhibitors were tested by competitive assay against benzoyl-tyrosine-p-nitroanilide as a substrate. The hydrolytic reaction was carried out at 37 °C at pH ) 7.00 with 50 mM Tris buffer and 0.02 M CaCl2. Inhibitor stock solutions in DMSO/H2O (1:10) were prepared with concentrations ranging from 6.02 to 0.0602 mM. A stock enzyme solution of 0.2 mg/ mL was made in 1 mM HCl solution. A series of substrate concentrations (dissolved in 36.6% DMSO and 63.4% methanol) ranging from 0.043 to 0.43 mM was prepared. The assay was performed by adding 700 µL of Tris buffer to a 1.5 mL Epindorph tube followed by 100 µL of inhibitor and 100 µL of enzyme, and these were incubated for 10 min at 37 °C. Then 100 µL of substrate was added to the reaction followed by incubation for 20 min at 37 °C. The reaction was stopped by adding 200 µL of 30% acetic acid solution. The progress of the reaction was followed by monitoring the appearance of the absorption band of p-nitroaniline at 410 nm. Ki values for reversible competitive inhibitors were estimated by the method of Lineweaver and Burk. Data were fitted to the best straight line by the least-squares procedure. Trypsin, type II-S (EC 3.4.21.4) from porcine pancreas, was purchased from Sigma. The inhibition constants were determined exactly as was done for chymotrypsin except that the substrate was benzoyl-arginine-p-nitroanilide dissolved in DMF. Elastase, type II-A (EC 3.4.21.36) from porcine pancreas, was purchased from Sigma. The hydrolytic reaction was carried out at 37 °C, standard pH 7.5, with 50 mM phosphate buffer, 0.2 M NaCl, and the progress of the reaction was followed by monitoring the appearance of the absorption band of p-nitroaniline at 410 nm (substrate ) N-succinyl-(Ala)3-p-nitroanilide). Leucine aminopeptidase, type VI (EC 3.4.11.2) from porcine kidney microsomes, was purchased from Sigma. The hydrolytic

Smoum et al.

reaction was carried out at 37 °C, standard pH 7.5, with 50 mM phosphate buffer, 0.02 M CaCl2, 10 vol % DMF, and the progress of the reaction was followed by monitoring the appearance of the absorption band of p-nitroaniline at 410 nm (substrate ) L-leucine-p-nitroanilide). In Vitro Evaluation of the Protective Effect of the Succinylated Chitosan-Phenylboronic Acid, ChitosanGlycylglycine-Phenylboronic Acid, and Chitosan-Pentaglycine-Phenylboronic Acid Conjugates toward Serine Proteases and Leucine Aminopeptidase. One to ten milligrams of the polymer-inhibitor conjugates was hydrated in 1 mL of 80 mM TBS buffer (Tris-HCl buffered saline), pH 6.8. Thereafter, trypsin (28.4 units), chymotrypsin (1 unit), elastase (0.4 units/mL), or leucine aminopeptidase (0.12 units/mL) dissolved in 100 µL of 80 mM TBS, pH 6.8, was added, and the mixture incubated for 1 h at room temperature. After addition of the corresponding substrates, each dissolved in a certain solvent as mentioned above, the reaction mixture was incubated for 20 min. The reaction mixture was stopped by adding 200 µL of 30% acetic acid solution. The absorbance (410 nM) caused by the formation of p-nitroanilide was recorded. In Vitro Evaluation of the Protective Effect of the Succinylated Chitosan-Phenylboronic Acid, ChitosanGlycylglycine-Phenylboronic Acid, and Chitosan-Pentaglycine-Phenylboronic Acid Conjugates toward Trypsin, Elastase, and Chymotrypsin in the presence of Chitosanase. The inhibitory activity of succinylated chitosan-phenylboronic acid, chitosan-glycylglycine-phenylboronic acid, and chitosan-pentaglycine-phenylboronic acid conjugates toward trypsin, elastase, and chymotrypsin was determined by the following enzyme assays. One milligram of the polymer conjugate was hydrated in 975 µL of 50 mM acetate buffer (pH 5.5). The solution was incubated for 5 min at 37 °C. Then, 50 µL of a chitosanase solution (chitosan N-acetylglucosaminohydrolase; EC 3.2.1.132 from Streptomyces species, 0.1 units dissolved in 55 mM acetate buffer, pH 5.5) was added, and the mixture was incubated for 1 h at 37 °C. Thereafter, 100 µL of the enzyme solution (trypsin (8.88 units/mL), elastase (0.4 units/mL), or chymotrypsin (156 units/mL)) in 50 mM acetate buffer, pH 5.5, was added, and the mixture was incubated at room temperature for 30 min. Following incubation, 100 µL (0.1 M solution) of the substrate (benzoyl-arginine-p-nitroanilide in DMF for trypsin, N-succinyl-Ala-Ala-Ala-p-nitroanilide in 20% DMF/ H2O for elastase, or benzoyl-tyrosin-p-nitroanilide in methanol/ DMSO for chymotrypsin) was added, and the enzymatic reaction was allowed to proceed at room temperature for 40 min for trypsin and 20 min for both elastase and chymotrypsin. The enzymatic activity was stopped by adding 200 µL of 30% acetic acid solution. The progress of the reaction was followed by monitoring the appearance of the absorption band of pnitroaniline at 405 nm. The controls used were the buffer and the unmodified chitosan. Evaluation of the Protective Effect for Calcitonin. Salmon calcitonin solutions were freshly prepared prior to experiments conducted at a concentration of 1 mg/mL. Three milligrams of succinylated chitosan-phenylboronic acid, chitosan-glycylglycine-phenylboronic acid, and chitosan-pentaglycine-phenylboronic acid conjugates were each dissolved in 950 µL of 50 mM acetate buffer, pH 5.5. Each solution was incubated for 5 min at 37 °C. Then, 50 µL of a chitosanase solution was added, and the solution was incubated for 1 h at 37 °C. Thereafter, 100 µL of a calcitonin solution (2 mg of salmon calcitonin dissolved in 100 µL of 50 mM acetate buffer, pH 5.5) was added, and the solution was incubated at room temperature for 20 min. Following this, 100 µL of the same buffer but containing trypsin (8.88 mg/mL) was added. Immediately after the addition of the enzyme solution, aliquots

Bioconjugate Chem., Vol. 17, No. 4, 2006 1003

Chitosan−Pentaglycine−Phenylboronic Acid Conjugate Scheme 1a

a

Reagents and conditions: (i) succinic anhydride, AcOH, H2O, MeOH; (ii) 1% aq NaOH, room temp, 2 h.

of 60 µL were withdrawn and diluted with the same volume of 1% trifluoroacetic acid (TFA) used as stop solution to avoid any enzymatic degradation of the peptide. These first samples were used as reference values (time point zero). After that, aliquots of 60 µL were withdrawn at 1, 5, 10, 20, 30, and 60 min, and the enzymatic degradation was terminated as described above. All samples were centrifuged to remove any remaining polymer contents. For determining the remaining undigested calcitonin, 20 µL of the supernatant fluid was directly injected for HPLC analysis. Samples prepared in the same way but without adding chitosanase were used as controls. In addition, samples prepared in the same way but containing 3 mg of unmodified chitosan instead of chitosan-inhibitor conjugates served as controls. HPLC Analytical Method. Salmon calcitonin (sCT) was analyzed by means of a validated HPLC method. HPLC Computer Controlled Waters Chromatography workstation with the following components was used: control 600, water pump 600, detector 996 photodiode array, and Millenium32 chromatography software. Room temperature was maintained for the column, and chromatographic separations were performed on a Waters RP Symmetry C18 4.6 mm × 250 mm column. Twenty microliters of sample was injected into the column, and samples were analyzed by a reversed phase HPLC method. The mobile phase consisted of 0.1% v/v TFA/water (A) and 0.1% v/v TFA/acetonitrile (B). The gradient conditions were 2035% B for 10 min, 35-37% B from 10 to 20 min, and 3720% B from 20 to 25 min at a flow rate of 1 mL/min. The detection was achieved at a wavelength of 210 nm. Concentrations of sCT were quantified from integrated peak areas and calculated by interpolation from the standard curve.

RESULTS Synthesis and Structural Analysis of Succinylated Chitosan-Phenylboronic Acid Conjugates. New chitosan derivatives were synthesized by direct reductive N-alkylation of chitosan with 4-formylphenylboronic acid, 2. Scheme 1 shows the preparation of chitosan-phenylboronic acid conjugates 3 by reductive N-alkylation using sodium borohydride (NaBH4). The degree of N-substitution could be controlled by increasing the amount of the aldehyde 2 in the reaction mixture (Table 1). The reactivity of 2 was found to vary in the range of 47-80%, which was caused by the simultaneous reduction of some of the aldehyde groups of 2 under the acidic conditions. The degree of N-substitution was calculated from the ratio of C% to N% in elemental analysis (17). In the FTIR spectra of chitosan and chitosan derivatives, the characteristic peaks of the amine NH vibration deformation appeared at 1595 cm-1 for chitosan, whereas it disappears in chitosan derivatives caused by the formation of NH-CH2 band at C-2 in chitosan and a new peak at a higher wavelength (1611.31 cm-1) due to C-C stretches in the aromatic ring. Characteristic absorption bands of para-

Table 2. Chemical Structures of the Succinylated Conjugates 4 functional group (DS) conjugates

-boronic compda

-Sucb

-NH2c

-NHAcd

1 2 3 4 5

0.469 0.512 0.530 0.560 0.580

0.34 0.32 0.32 0.32 0.32

0.041 0.018 0.000 0.000 0.000

0.15 0.15 0.15 0.15 0.15

a DS (degree of substitution) of phenylboronic acid residues. b DS of succinate residues. c Free amine residues. d Free acetylated amine residues.

substituted benzene appeared at 650.47, 816.05, 1515.33, 1560.80, and 1611.31. Water-soluble material was only obtained moderately at high degree of substitution (DS ) 79.9%). However, the other chitosan-boronic acid conjugates 3 were insoluble in neutral water and thus would not be useful for biological evaluation. To improve the solubility, the remaining amino groups of the conjugates 3 were transformed by succinylation with succinic anhydride to give conjugates 4 in 90-100% yields. The chemical structures of the succinylated conjugates 4 are summarized in Table 2. The degree of substitution for most compounds was between 32% and 34%. Despite using a large excess of succinic anhydride, some unreactive amino groups still remained in several conjugates. High-field 1H NMR spectra also indicated that no succinylation had occurred at the N-glycosylation sites (δ H-1 of GclN-Neu5Ac). All succinylated products 4 were soluble in water. Synthesis and Structural Analysis of the ChitosanSpacer-Phenylboronic Acid Conjugates (Spacer ) Glycylglycine or Pentaglycine). As shown in Scheme 2, 4-formylphenylboronic acid was attached to the spacer, glycylglycine or pentaglycine, by reductive N-alkylation using sodium borohydride (NaBH4). The resulting spacer-phenylboronic acid conjugates were covalently coupled to the amino group of chitosan using EDAC, a water-soluble carbodiimide, Scheme 3. Two sets of conjugates were prepared (Figures 1 and 2); each consisted of different amounts of conjugated glycylglycine/ pentaglycine-phenylboronic acids (Tables 3 and 4). The degree of N-substitution was determined by 1H NMR and elemental analysis. As can be seen, there was a good correlation between the data obtained by the two methods. Conjugation was confirmed by the existence of an amide band at 1653.77 cm-1 from the FTIR spectrum of chitosan derivatives. The characteristic peaks of amine NH vibration deformation at 1595 cm-1 found in chitosan disappeared in the chitosan derivatives. Characteristic absorption bands of para-substituted benzene ring appeared at 653.53, 898.09, and 1560.03. All the conjugates prepared were soluble in water. The inhibitory efficacy of the 4-formylphenylboronic acid, glycylglycine-phenylboronic acid, and pentaglycine-phenylboronic acid as well as their corresponding chitosan conjugates (succinylated chitosan-phenylboronic acid, chitosan-glycyl-

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Scheme 2. Preparation of the Spacer-Boronic Acid Conjugates

Scheme 3. Preparation of the Chitosan-Spacer-Boronic Acid Conjugates

Table 3. Preparation of Chitosan-Glycylglycine-Phenylboronic Acid Conjugates entry

conjugate

DSa

recoveryb (wt %, g/g)

solubility in H2O

elemental analysis C H N

1 2 3 4 5

0.2 0.4 0.6 0.8 1.0

23.18 25.70 28.17 29.73 30.50

80.8 78.6 79.3 76.2 76.5

moderate yes yes yes yes

40.61 39.24 40.14 40.03 39.29

6.63 6.69 6.76 6.73 6.71

6.75 6.61 6.48 6.40 6.36

a

DS was determined from the peak area of phenyl protons (δ 7.0-7.4) and H-2 of GlcN and N-alkylated GlcN residues (δ 3.2) and from elemental analysis. b Recovery (%) was obtained by dividing the obtained product weight by the weight of the chitosan: Recovery (%) ) [wt of product (g)/ wt of DAC 85%] × 100.

Table 4. Preparation of Chitosan-Pentaglycine-Phenylboronic Acid Conjugate entry

conjugate

DSa

1 2 3 4

0.4 0.6 0.8 1.0

26.06 28.20 29.00 43.75

recoveryb solubility (wt %, g/g) in H2O 72.8% 76.4% 72.7% 78.6%

yes yes yes yes

elemental analysis C H N 40.53 40.58 40.38 40.69

6.64 6.52 6.48 6.33

9.42 9.43 9.48 10.46

a DS was determined from the peak area of phenyl protons (δ 7.0-7.4) and H-2 of GlcN and N-alkylated GlcN residues (δ 3.2) and from elemental analysis. b Recovery (%) was obtained by dividing the obtained product weight by the weight of the chitosan: Recovery (%) ) [wt of product (g)/ wt of DAC 85%] × 100.

Table 5. Inhibition Constants (Ki in mM) for the 4-Formylphenylboronic Acid, Glycylglycine-Phenylboronic Acid, and Pentaglycine-Phenylboronic Acid as Measured with Chymotrypsin, Trypsin, Elastase, and Leucine Aminopeptidase

compound

Figure 1. Structure of chitosan-glycylglycine-phenylboronic acid conjugate.

Figure 2. Structure of chitosan-pentaglycine-phenylboronic acid conjugate.

glycine-phenylboronic acid, and chitosan-pentaglycinephenylboronic acid) toward serine proteases and leucine aminopeptidase was determined. In addition, the protective effect of these conjugates for salmon calcitonin toward the luminally secreted serine protease trypsin was evaluated. Enzyme Inhibition Studies. Enzyme inhibition studies were carried out with trypsin, R-chymotrypsin, elastase, and leucine aminopeptidase N. Inhibitory Efficacy of 4-Formylphenylboronic Acid, Glycylglycine-Phenylboronic Acid, and Pentaglycine-Phenylboronic Acid Conjugates. 4-Formylphenylboronic acid was chosen to

4-formylphenylboronic acid glycylglycine-phenylboronic acid pentaglycine-phenylboronic acid

leucine chymoaminotrypsin trypsin elastase peptidase 0.4 6 5

0.8 3 3

0.6 2 3

4 >10 8

be conjugated to chitosan because it is a simple molecule with an aldehyde group that can be conjugated to the amino group of the chitosan by reductive N-alkylation. This compound was checked for its ability to inhibit the serine proteases and leucine aminopeptidase. It was found that 4-formylphenylboronic acid is capable of inhibiting trypsin, chymotrypsin, elastase, and leucine aminopeptidase in a moderate range (Table 5). 4-Formylphenylboronic acid was then attached to two spacers, glycylglycine and pentaglycine. The influence of the spacers on the inhibition ability of 4-formylphenylboronic acid was checked. It was found that both the glycylglycine-phenylboronic acid and pentaglycine-phenylboronic acid were still able to inhibit trypsin, chymotrypsin, elastase, and leucine aminopeptidase (Table 5). However, the activities of these conjugates were an order of magnitude less than the original 4-formylphenylboronic acid. Inhibitory Efficacy of Chitosan-Phenylboronic Acid Conjugates with and without Spacers. The chitosan-phenylboronic acid conjugates without any spacer were not soluble in water, DMSO, or methanol; however, they were soluble in 1% acetic

Chitosan−Pentaglycine−Phenylboronic Acid Conjugate

Bioconjugate Chem., Vol. 17, No. 4, 2006 1005

Figure 3. The assumed cleavage of chitosan-spacer-phenylboronic acid conjugate by the chitosanase enzyme (spacer ) none, glycylglycine, or pentaglycine).

acid/H2O. On the other hand, their succinylated derivatives were soluble in water, and therefore, they were used for biological evaluation. All the succinylated conjugates with different degrees of substitutions (Table 2) were checked for protease inhibition against the serine proteases trypsin, chymotrypsin, and elastase at concentrations up to 10 mg/mL. All conjugates with different degrees of substitution did not inhibit the serine proteases at any concentration. In addition, the chitosan-glycylglycine-phenylboronic acid and the chitosan-pentaglycine-phenylboronic acid conjugates at all degrees of substitutions and at concentrations up to 10 mg/mL were tested for their ability to inhibit serine proteases. In the same manner, all these conjugates were not capable of inhibiting the enzymes. Inhibitory Efficacy of Chitosan Conjugates with and without Spacers in the Presence of Chitosanase: A Proof of Concept. All the chitosan conjugates that contained phenylboronic acid without and with spacers did not inhibit the serine proteases. This may be because the conjugates were so bulky that the phenylboronic acid compound was not accessible to the active site of the enzyme even with the use of short and long spacers. As mentioned before, chitosan is hydrolyzed by bacterial glycosidases in the colon (14). For the conjugated polymers prepared in this paper, this hydrolysis would allow the release of monomers, dimers, and oligomers of chitosan that are conjugated to 4-formylphenylboronic acid with or without spacers. These smaller conjugates may inhibit in turn the serine proteases and leucine aminopeptidase. Therefore, this led to the idea of using an enzyme that would be able to hydrolyze the polymer conjugate into monomers, dimers, and oligomers similarly as would the bacterial enzymes in the colon. In this context, the enzyme chitosanase was chosen. Chitosanase is responsible for the endohydrolysis of β-1,4linkages between GlcNAc-GlcN and GlcN-GlcN bonds in a partly acetylated chitosan (18) (Figure 3). Chitosanase is used as a model for glycosidases that can hydrolyze chitosan in the colon. The polymers were incubated with chitosanase for 1 h, a time that was calibrated for the complete hydrolysis of chitosan into monomers, dimers, and oligomers. This was followed by the addition of the required enzyme (trypsin, chymotrypsin, elastase, or leucine aminopeptidase) and incubation of the mixture for another 1 h, after which the substrate was added. The results showed that only the conjugates with the pentaglycine spacer were able to inhibit trypsin and elastase, Figure 4. In these experiments, the buffer and the unmodified chitosan were used as controls. The inhibition increases with increasing the degree of substitution of the polymer conjugate. The polymer conjugate with the highest degree of substitution

Figure 4. Effect of the buffer, chitosan, and the chitosan-pentaglycine-boronic acid conjugates with different degrees of substitutions on the relative activity of elastase (top) and trypsin (bottom).

(43%) demonstrated a strong inhibitory activity toward trypsin resulting in a significant decrease in the degradation of the trypsin substrate N-R-benzoyl-L-arginine-p-nitroanilide. After 40 min of enzymatic reaction, only 50% of the substrate was digested in the presence of 1 mg of chitosan conjugate, whereas at this reference point, 100% of the substrate was degraded in the presence of the same concentration of unmodified chitosan.

1006 Bioconjugate Chem., Vol. 17, No. 4, 2006

Figure 5. The degradation of sCT in the presence of chitosanase and (2) 1 mg of chitosan(Gly)5B(OH)2 or (9) 3 mg of chitosan(Gly)5B(OH)2 or (b) 3 mg of chitosan(Gly)5B(OH)2 without chitosanase (control). Shown are the mean of 2 different measurements.

Also, elastase was inhibited by the same conjugate, but the effect displayed by the decrease in the degradation of the substrate succinyl-(alanyl)3-p-nitroanalide was not as pronounced as in the case of trypsin. The chitosan-spacer-phenylboronic acid conjugates were also evaluated with regard to their inhibitory activity toward R-chymotrypsin and leucine aminopeptidase in the presence of chitosanase, but no significant inhibition of the enzymes was observed. Protective Effect toward Calcitonin. The chitosan-pentaglycine-phenylboronic acid conjugate with the highest degree of substitution (DS ) 43%), which inhibited 50% of trypsin and 30% of elastase at 1 mg/ml concentration, was evaluated for a protective effect on the enzymatic degradation of calcitonin. After 10 min incubation with the proteases, only 8.4% of undegraded salmon calcitonin remained in a solution of chitosan-pentaglycine-boronic acid conjugate without the use of the chitosanase. With the use of chitosanase, only 13% undegraded salmon calcitonin remained in the presence of 1 mg of chitosan-pentaglycine-boronic acid. But by increasing the amount of the conjugate to 3 mg in the presence of chitosanase, 40% of undegraded calcitonin remained (Figure 5).

DISCUSSION In the present study, novel boronated chitosan conjugates were prepared and tested for their ability to potentially serve as oral delivery systems of calcitonin. Chitosan was selected due to its mucoadhesive properties (19), as well as strong permeation enhancing capabilities for protein drugs (20). It was shown that its mucoadhessiveness was maintained even after conjugation with enzyme inhibitors (21), allowing it to function in close proximity to the intestinal wall, while providing a continuous protection to its protein drug cargo. The combined use of mucoadhesive polymers and enzyme inhibitors could be interesting (22). However, there is always a risk of losing the protease inhibitor along the journey in the lumen of the gut. This can be resolved by their covalent attachment to polymeric drug carrier matrices, allowing them safety as long as the platform retains its integrity. Chitosan derivatives exhibiting a better solubility, stronger mucoadhesive capabilities, and enzyme inhibitory properties toward luminal secreted proteases and brush border membrane bound peptidases could be generated. These unique features make chitosan and in particular its derivatives an interesting excipient for the preoral administration of peptide drug (23). 4-Formylphenylboronic acid was found to be a moderate inhibitor of serine proteases. It was conjugated through its

Smoum et al.

aldehydic group to chitosan by reductive N-alkylation. NSuccinylation of the prepared conjugates gave water-soluble succinylated chitosan-phenylboronic acid conjugates. In our study, succinylated conjugates were unable to inhibit serine proteases and leucine peptidase, probably due to the bulkiness of the polymer conjugate, so the inhibitor 4-formylphenylboronic acid was not accessible enough to the protease to inhibit it, even with the high degree of N-substitution. However, BernkopSchnu¨rch and co-workers synthesized a series of mucoadhesive polymers that displayed strong protective effects toward all pancreatic proteases (24). In their study, the serine protease inhibitors antipain, chymostatin, and elastatinal were covalently linked to chitosan together with EDTA. These polymer conjugates showed a strong inhibitory activity toward trypsin, chymotrypsin, elastase, carboxypeptidases A and B, and aminopeptidase N. To increase the accessibility of the 4-formylphenylboronic acid to the enzyme, we suggested using spacers. Two kinds of spacers were chosen, glycylglycine and pentaglycine spacers. The 4-formylphenylboronic acid-spacer conjugates inhibited serine proteases and leucine aminopeptidase moderately. However, after conjugating the spacer-phenylboronic acids to chitosan, no inhibition for the resulting polymer conjugates could be detected. The use of the longer pentaglycine spacer was thought to improve the inhibitory effect. Yet, inhibition studies revealed opposite results. Indeed, the inhibitory activity of chitosan-EDTA-ACE (ACE ) antipain, chymostatin, elastatinal) conjugate toward elastase was markedly weaker than that of poly(acrylate) derivative-elastatinal conjugates and carboxymethylcellulose-elastatinal conjugates (25). A reason for the comparably stronger inhibitory effect of these already established polymer conjugates can be seen in the additional use of a spacer, providing an easy accessibility of the immobilized inhibitor for the corresponding protease. Moreover, Marschu¨tz and co-workers reported recently that the use of a small rigid C4 spacer was highly advantageous compared to a long and more flexible poly(ethylene glycol) spacer (26). In our study, neither the short nor the long spacers had any effect on increasing the accessibility of the boronic acid to the active site of the protease. To mimic the colonic enzyme cleavage of chitosan, chitosanase was used in our study. This enzyme is responsible for the endohydrolysis of β-1,4-linkages between GlcNAc-GlcN and GlcN-GlcN bonds in a partly acetylated chitosan (18). Surprisingly, it was observed in our study that only in the presence of chitosanase were the pentaglycine derivatives of the conjugates active in a ratio-dependent manner. At a concentration of 1 mg/mL of the chitosan-pentaglycinephenylboronic acid conjugate with a 43% degree of substitution, 50% inhibition for trypsin and 30% inhibition for elastase were observed. We concluded that the chitosanase used in our study hydrolyzed the chitosan conjugates into monomers, dimers, and oligomers containing conjugated pentaglycine-phenylboronic acids. The higher the relative amount of pentaglycine-phenylboronic acid in the polymer, the more the degradation products, the monomers, dimers, and oligomers, were obtained. This observation supports the assumption that the polymer bulkiness was the reason for the interference with its enzyme inhibition capability. The luminally secreted serine proteases seem to be the major cause for the digestion of salmon calcitonin (sCT). It has been shown that sCT is degraded by trypsin, R-chymotrypsin (27), and elastase (28). Although many attempts have been undertaken, the development of a commercially applicable oral delivery system for this peptide has failed so far (3). The use of mucoadhesive polymers is believed to provide a prolonged residence time of the drug at the absorption site, leading to an

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Chitosan−Pentaglycine−Phenylboronic Acid Conjugate

improved bioavailability (29). To ascertain a protection within the polymeric carrier matrix (28), chitosan-enzyme inhibitor conjugates were added. Similar to the results obtained with thiolated polymers (30), our study showed that the conjugate with the highest degree of substitution (43%) was able to reduce the digestion of sCT by trypsin only in the presence of the chitosanase enzyme. Therefore, these conjugates seem to be promising agents in protecting sCT from intestinal pancreatic serine proteases. This study provides a proof of concept for further research aimed at identifying potent boron-based protease inhibitors.

ACKNOWLEDGMENT The study was supported by a research grant from the Israeli Science Foundation, Grant 663/ 99-2, and a Scholarship grant (R.S.) from the Ministry of Science and Technology. Morris Srebnik and Abraham Rubinstein are affiliated with the David R. Bloom Center of Pharmacy.

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