Pharmaceutical compositions comprising polymeric binders with non-hydrolysable covalent bonds and their use in treating celiac disease

Abstract

A pharmaceutical composition comprising a polymeric binder including a high molecular weight synthetic polymer having a backbone constituted of non hydrolysable covalent bonds, said polymer being able to form electrostatic bonds at a pH lower than the isoelectric point of gluten and peptides derived from the degradation of gluten, and being able to bind to gluten or peptides derived from the degradation of gluten in the gastrointestinal tract, and a pharmaceutically acceptable carrier. Methods of using the polymeric binder for binding gluten or a peptide derived from the degradation of gluten, for decreasing the degradation of gluten into toxic peptides or for decreasing interaction of gluten or peptides derived from the degradation of gluten with the gastrointestinal mucosa.

Claims

1. Food comprising a pharmaceutically effective amount of a polymeric binder including a high molecular weight synthetic linear copolymer, the synthetic linear copolymer comprising a linear copolymer of hydroxyethyl methacrylate (HEMA) and 4-styrene sulfonic acid or a salt thereof, wherein said linear copolymer has a molar percentage ratio of HEMA:4-styrene sulfonic acid or a salt thereof from between about 82.4:17.6 mol % to about 28:72 mol %, and a processed food.

2. The food of claim 1, wherein the food is a gluten-containing food.

3. The food of claim 2, wherein the food is bread.

4. The food of claim 1, wherein the synthetic linear copolymer comprises a copolymer of HEMA and SStNa hydrate.

5. The food of claim 4, wherein the copolymer of HEMA and SStNa is linear HEMA/SStNa, wherein the HEMA/SStNa ratio is 51.5/48.5 mol %.

6. The food of claim 4, wherein the copolymer of HEMA and SStNa is linear HEMA/SStNa, wherein the HEMA/SStNa ratio is 43/57 mol %.

7. The food of claim 4, wherein the synthetic linear copolymer of HEMA and SStNa contains about 50% SStNa.

8. The food of claim 1, wherein the synthetic linear copolymer has a backbone constituted of non-hydrolysable covalent bonds.

9. The food of claim 1, wherein the synthetic linear copolymer is able to form electrostatic bonds at a pH lower than the isoelectric point of gluten and peptides derived from the degradation of gluten.

10. The food of claim 1, wherein the synthetic linear copolymer is able to form hydrophobic interactions with gluten or peptides derived from the degradation of gluten.

11. The food of claim 1, wherein the synthetic linear copolymer is able to form hydrogen bonds.

12. The food of claim 1, wherein the synthetic linear copolymer is able to specifically bind to gluten or peptides derived from the degradation of gluten in the gastrointestinal tract.

13. The food of claim 1, wherein the synthetic linear copolymer is able to bind to gluten or peptides derived from the degradation of gluten in the intestinal tract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the appended drawings:

(2) FIG. 1 presents the chemical structures of linear and multifunctional ATRP initiators used to synthesize polymers described herein. (i) PEG-dibromo macroinitiator; (ii) 1,2,3,4,6-penta-O-isobutyryl bromide-R-D-Glucose; (iii) Octadeca-O-isobutyryl bromide-R-cyclodextrin; (iv) Octa-O-isobutyryl bromide-sucrose;

(3) FIG. 2 presents the SOS-PAGE of the binding of albumin and α-gliadin with poly(HEMA-co-SStNa) (Example 10) at pH 6.8 in triplicate: (A) protein standards; (8) albumin and a-gliadin mixture; (C) mixture of albumin (40 mg/L), α-gliadin (40 mg/L) and poly(HEMA-co-SStNa) (160 mg/L);

(4) FIG. 3 is a binding profile of linear poly(HEMA-co-SStNa) to gliadin, albumin, and casein at pH 1.2 and 6.8, wherein each point corresponds to the polymer of each of Examples 5 to 10 and 12-13;

(5) FIG. 4 is a binding profile of linear poly(HEMA-co-SPMAK) to gliadin and albumin at pH 1.2 and 6.8 wherein each point corresponds to the polymer of each of Examples 17 to 21;

(6) FIG. 5 graphically presents the polymer structure effect on the binding of gliadin at neutral pH (SStNa=25-31 mol %; see Examples 9, 14, 15 and 16);

(7) FIG. 6 graphically presents the polymer structure effect on the binding of gliadin at neutral pH (SPMAK=16-19 mol %; see Examples 13, 22, 23 and 24);

(8) FIG. 7 presents the effect of a polymer of the invention on gliadin digestion under simulated intestinal conditions. Comparative HPLC profiles of gliadin digested with pepsin, trypsin and chymotrypsin (PTC) in absence (a) and presence (b) of polymer. Chromatogram (c) corresponds to intact α-gliadin;

(9) FIG. 8 presents the variation of the transepithelial electric resistance (TEER) of a Caco-2 monolayer following incubation with solutions of PEG (Mn: 35,000; open circles), PVP (Mw: 58,000; closed squares), poly(HEMA-co-SStNa) (Example 10; open triangles) and complete medium (closed stars) as a control. Cells were maintained in DMEM cell culture media supplemented with 10% FBS. The polymer concentration was fixed at 1 g/L; and

(10) FIG. 9 presents a binding profile of two linear poly(HEMA-co-SStNa) containing about 50% SStNa and having two different molecular weights (Examples 10 and 11) to gliadin and albumin at pH 1.2 and 6.8.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(11) Materials

(12) α-Gliadin was kindly supplied by the Institut National de la Recherche Agronomique, (Nantes, France). It was purified from soft wheat as described by Popineau et al. (16-21). Briefly, after extraction of crude gliadin from gluten (isolated from flour), gliadin subgroups were separated and purified successively by ion exchange chromatography, size exclusion chromatography and finally hydrophobic interaction chromatography.

(13) Bovine albumin was purchased from Serological Proteins (Kankakee, Ill.). α-Casein (from bovine milk), SStNa, HEMA, SPMAK, R-D-glucose, α-cyclodextrin hydrate, sucrose (98%), poly(ethylene glycol) (PEG) (M.sub.n 2000), 2-bromoisobutyryl bromide, copper bromide Cu(I)Br and 2,2′dipyridyl were all purchased from Sigma-Aldrich (St Louis, Mo.) and used as received. Eppendorff tubes, pipette tips and 96-well plates (Maximum Recovery) were provided from Axygen Scientific (Union City, Calif.).

(14) Synthesis of the Initiators

(15) Atom transfer radical polymerization (ATRP) initiators (FIG. 1) were prepared from PEG, R-D-glucose, sucrose and a-cyclodextrin. The bromide functionalization of the last three molecules was achieved by the approach described by Stenzel-Rosenbaum and co-workers (22).

(16) The present invention is illustrated in further details by the following non-limiting examples.

Example 1

Synthesis of PEG Dibromomacroinitiator (i)

(17) A solution of HO-PEG-OH (M.sub.n 2000, 10 g, 5 mmol) and triethylamine (10 g, 0.1 mol) in 70 mL of anhydrous toluene was slightly cooled in an ice-water bath. Then, 2-bromoisobutyryl bromide (4.91 mL, 0.04 mol) was slowly added to the reaction mixture. The solution was warmed to room temperature and stirred for 48 h. The mixture was filtered, half of the solvent was evaporated, and the PEG macroinitiator was precipitated in cold diethyl ether (FIG. 1 (i)).

(18) Yield: 90%, after precipitation. White solid. .sup.1H NMR (δ, ppm, CDCl.sub.3): 3.50 (188H), 1.80 (12H, s).

Example 2

Synthesis of 1,2,3,4,6-Penta-O-isobutyryl bromide-R-D-glucose (ii)

(19) 2-bromoisobutyryl bromide (50 g, 0.22 mol) was slowly added to a solution of R-D-glucose (5.0 g, 0.028 mol) in an anhydrous mixture of chloroform (100 mL) and pyridine (50 mL). The solution was refluxed for 3 h while maintaining a dry atmosphere and then stirred at room temperature for a further 12 h. It was then washed successively with ice-cold water, NaOH (0.1 M), and water and dried over anhydrous MgSO.sub.4. The crude product was recrystallized from methanol to yield white crystals (FIG. 1 (ii)).

(20) Yield: 70%. .sup.1H NMR (CDCl.sub.3): 1.85-2.04 (m, 30H, H-7), 6.42 (d, 1H, H-1), 5.25 (dd, 1H, H-2), 5.69 (t, 1H, H-3), 5.35 (t, 1H, H-4), 4.38 (m, 3H, H-5/6).

Example 3

Synthesis of Octadeca-O-isobutyryl Bromide-R-cyclodextrin (iii)

(21) Octadeca-O-isobutyryl bromide-R-cyclodextrin was synthesized by the slow addition of 2-bromoisobutyryl bromide (50 g, 0.22 mol) to a solution of R-cyclodextrin (5.0 g, 0.005 mol) in anhydrous pyridine (150 mL). The solution was stirred for 24 h under a dry atmosphere at room temperature. It was then washed with ice-cold water, NaOH (0.1 M), and water, respectively, prior to drying over anhydrous MgSO.sub.4. The crude product was recrystallized from methanol/H.sub.2O (3:1, v/v) to yield white crystals (FIG. 1 (iii)).

(22) Yield: 55%. .sup.1H NMR (CDCl.sub.3): 1.95 (m, 108H, H-7), 5.84 (d, 12H, H-1), 4.46 (dd, 6H, H-2), 5.7 (m, 6H, H-3), 5.13/5.38 (t/dd, 6H, H-4), 4.78 (dd, 6H, H-5), 4.45 (m, 6H, H-6).

Example 4

Synthesis of Octa-O-isobutyryl bromide-sucrose (iv)

(23) Octa-O-isobutyryl bromide sucrose was synthesized by the slow addition of 2-bromoisobutyryl bromide (50 g, 0.22 mol) to a solution of sucrose (5.0 g, 0.014 mol) in anhydrous pyridine (150 mL). The solution was stirred for 24 h under a dry atmosphere at room temperature. It was then washed with ice-cold water, NaOH (0.1 M), and water, prior to drying over anhydrous MgSO.sub.4. The crude product was recrystallized from methanol/H.sub.2O (3:1 v/v) to yield white crystals (FIG. 1 (vi)).

(24) Yield: 50%. .sup.1H NMR (CDCl.sub.3): 1.99 (m, 48H, H-7), 4.15 (d, 1H, H-5′), 4.46 (m, 5H, H-6′/1′/5), 4.68 (dt, 2H, H-6), 4.81 (d, 1H, H-3′), 5.13 (dd, 1H, H-2), 5.38 (t, 1H, H-4′), 5.67 (t, 1H, H-4), 5.76 (t, 1H, H-3), 5.85 (d, 1H, H-1).

Example 5

Synthesis of Linear Hydroxyethyl Methacrylate (HEMA)/4-Styrene Sulfonic Acid Sodium Salt Hydrate (SStNA) Copolymer (93.5/6.5 mol % After Purification)

(25) The ATRP initiator i (FIG. 1) (50 mg), SStNa (0.375 g) and HEMA (7.12 g) were dissolved in 46 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (20.28 mg), Cu(I)Br (7.2 mg) and Cu(II)Br.sub.2 (3.35 mg) were then added under stirring at 20° C., After 24 h, the solution was exposed to air and the dark-brown solution turned to blue, indicating oxidation of Cu(I) to Cu(II). The polymer was purified by passing the methanol/water solution through a silica gel column which removed the Cu(II) catalyst. The polymers were dialyzed (Spectra/Por™ no. 1, MW cutoff 6000-8000 Spectrum Laboratories, Rancho Dominguez, Calif.) against water for 48 h and then freeze-dried until use. M.sub.w=318700 g/mol; M.sub.w/M.sub.n=2.54.

Example 6

Synthesis of Linear Hydroxyethyl Methacrylate/4-Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (90.3/9.7 mol % After Purification)

(26) The ATRP initiator i (FIG. 1) (50 mg), SStNa (0.375 g) and HEMA (7.12 g) were dissolved in 46 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (21.84 mg), Cu(I)Br (7.2 mg) and Cu(I)Br.sub.2 (4.48 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=331 528, M.sub.w/M.sub.n=2.9

Example 7

Synthesis of Linear Hydroxyethyl Methacrylate/4-Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (87.8/12.2 mol % After Purification)

(27) The ATRP initiator i (FIG. 1) (50 mg), SStNa (0.75 g) and HEMA (6.747 g) were dissolved in 46 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=283 600 g/mol; M.sub.w/M.sub.n=2.57.

Example 8

Synthesis of Linear Hydroxyethyl Methacrylate/4-Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (82.4/17.6 mol % After Purification)

(28) The ATRP initiator i (FIG. 1) (50 mg), SStNa (1.125 g) and HEMA (6.426 g) were dissolved in 46 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Cl (5 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=275 500 g/mol; M.sub.w/M.sub.n=2.5.

Example 9

Synthesis of Linear Hydroxyethyl Methacrylate/4.Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (69/31 mol % After Purification)

(29) The ATRP initiator i (FIG. 1) (50 mg), SStNa (1.5 g) and HEMA (5.99 g) were dissolved in 46 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Cl (5 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=NA; M.sub.w/M.sub.n=NA. Mn.sub.(NMR)=58 100 g/mol.

Example 10

Synthesis of Linear Hydroxyethyl Methacrylate/4-Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (51.5/48.5 mol % After Purification)

(30) The ATRP initiator i (FIG. 1) (50 mg), SStNa (3.2 g) and HEMA (3.95 g) were dissolved in 46 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=122 000 g/mol; M.sub.w/M.sub.n=2.23.

Example 11

Synthesis of Linear Hydroxyethyl Methacrylate/4.Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (43/57 mol % After Purification)

(31) The ATRP initiator i (FIG. 1) (50 mg), SStNa (2.4 g) and HEMA (1 g) were dissolved in 23 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. Mn.sub.(NMR)=55 000 g/mol.

Example 12

Synthesis of Linear Hydroxyethyl Methacrylate/4-Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (28/72 mol % After Purification)

(32) The ATRP initiator i (FIG. 1) (50 mg), SStNa (4.8 g) and HEMA (1.975 g) were dissolved in 46 mL of a methanol/water (1/4) mixture and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=65 200 g/mol; M.sub.w/M.sub.n=1.95.

Example 13

Synthesis of Linear Poly(4-Styrene Sulfonic Acid Sodium Salt Hydrate)

(33) The ATRP initiator i (FIG. 1) (50 mg) and SStNa (6.4 g) were dissolved in 46 mL of water and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=NA; M.sub.w/M.sub.n=NA. M.sub.n (NMR)=20 000 g/mol.

Example 14

Synthesis of Linear Poly(4.Styrene Sulfonic Acid Sodium Salt Hydrate)

(34) The ATRP initiator i (FIG. 1) (50.3 mg) and SStNa (1.56 g) were dissolved in 20 mL of water and degassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=NA; M.sub.w/M.sub.n=NA. M.sub.n (NMR)=57 500 g/mol.

Example 15

Synthesis of 5-Arm Star Hydroxyethyl Methacrylate/4-Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (69/31 mol % After Purification)

(35) The ATRP initiator ii (FIG. 1) (142.6 mg), SStNa (1.55 g) and HEMA (4.616 g) were dissolved in 30 of a methanol/water (8/1) mixture and degassed under argon for 15 min. Bpy (230.75 ma) and Cu(I)Br (106 mg) were then added under stirring at 20° C. After 1 h of reaction, 10 mL of water were added and the solution was then maintained at room temperature for 24 h. The corresponding copolymer was finally purified as reported in Example 5. M.sub.w=85 000 g/mol; M.sub.w/M.sub.n=1.79.

Example 16

Synthesis of 8.Arm Star Hydroxyethyl Methacrylate/4.Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (75/25 mol % After Purification)

(36) The ATRP initiator iv (FIG. 1) (141 mg), SStNa (1.5 g) and HEMA (4.616 g) were dissolved in 30 mL of a methanol/water (8/1) mixture and degassed under argon for 15 min, Bpy (230.8 mg) and Cu(I)Br (106 mg) were then added under stirring at 20° C. After 1 h of reaction, 10 mL of water were added and the solution was then maintained at room temperature for 24 h. The corresponding copolymer was finally purified as reported in Example 5. M.sub.w=210 000 g/mol; M.sub.w/M.sub.n=2.03.

Example 17

Synthesis of 18.Arm Star Hydroxyethyl Methacrylate/4.Styrene Sulfonic Acid Sodium Salt Hydrate Copolymer (69/31 mol % After Purification)

(37) The ATRP initiator iii (FIG. 1) (153.5 mg), SStNa (1.5 g) and HEMA (4.62 g) were dissolved in 30 mL of a methanol/water (8/1) mixture and degassed under argon for 15 min. Bpy (230.75 mg) and Cu(I)Br (106 mg) were then added under stirring at 20° C. After 1 h of reaction, 10 mL of water were added and the solution was then maintained at room temperature for 24 h. The corresponding copolymer was finally purified as reported in Example 5. M.sub.w=206 000 g/mol; M.sub.w/M.sub.n=2.6.

Example 18

Synthesis of Linear Hydroxyethyl Methacrylate (HEMA)/Sulfopropyl Methacrylate Potassium Salt (SPMAK) Copolymer (86/14 mol % After Purification)

(38) The ATRP initiator i (FIG. 1) (100.3 mg), SPMAK (1.85 g) and HEMA (5.62 g) were dissolved in 30 mL of methanol and degassed under argon for 15 min. Bpy (31.96 mg) and Cu(I)Br (15.1 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=NA; M.sub.w/M.sub.n=NA. Mn.sub.(NMR)=66 500 g/mol.

Example 19

Synthesis of Linear Hydroxyethyl Methacrylate (HEMA)/Sulfopropyl Methacrylate Potassium Salt (SPMAK) Copolymer (83/17 mol % After Purification)

(39) The ATRP initiator i (FIG. 1) (102.1 mg), SPMAK (1.90 g) and HEMA (5.62 g) were dissolved in 30 mL of methanol and degassed under argon for 15 min. Bpy (31.24 mg) and Cu(I)Br (14.34 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=NA; M.sub.w/M.sub.n=NA. Mn.sub.(NMR)=84 000 g/mol.

Example 20

Synthesis of Linear Hydroxyethyl Methacrylate/Sulfopropyl Methacrylate Potassium Salt Copolymer (74/26 mol % After Purification)

(40) The ATRP initiator i (FIG. 1) (100.5 mg), SPMAK (3.75 g) and HEMA (5.622 g) were dissolved in 46 mL of a methanol/water (1/1) mixture and degassed under argon for 15 min. Bpy (32 mg) and Cu(I)Br (15.1 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=NA; M.sub.w/M.sub.n=NA. M.sub.n (NMR)=119 000 g/mol.

Example 21

Synthesis of Linear Hydroxyethyl Methacrylate/Sulfopropyl Methacrylate Potassium Salt Copolymer (45/55 mol % After Purification)

(41) The ATRP initiator i (FIG. 1) (100.7 mg), SPMAK (5.64 g) and HEMA (1.752 g) were dissolved in 46 mL of a methanol/water (1/1) mixture and degassed under argon for 15 min. Bpy (32 mg) and Cu(I)Br (15.1 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5 M.sub.w=NA; M.sub.w/M.sub.n=NA. Mn.sub.(NMR)=108 500 g/mol.

Example 22

Synthesis of Linear Poly(Sulfopropyl Methacrylate Potassium)

(42) The ATRP initiator i (FIG. 1) (100.7 mg) and SPMAK (7.5 g) were dissolved in 46 mL of a methanol/water (1/1) mixture and degassed under argon for 15 min. Bpy (32 mg) and Cu(I)Br (15.1 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=NA; M.sub.w/M.sub.n=NA. Mn.sub.(NMR)=120 000 g/mol.

Example 23

Synthesis of 5-Arm Star Hydroxyethyl Methacrylate/Sulfopropyl Methacrylate Potassium Copolymer (82.4/17.6 mol % After Purification)

(43) The ATRP initiator ii (FIG. 1) (143 mg), SPMAK (1.87 g) and HEMA (4.61 g) were dissolved in 60 mL of a methanol/water (8/1) mixture and degassed under argon for 15 min. Bpy (230.6 mg) and Cu(I)Br (108 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=161 000 g/mol; M.sub.w/M.sub.n=2.4.

Example 24

Synthesis of 8-Arm Star Hydroxyethyl Methacrylate/Sulfopropyl Methacrylate Potassium Copolymer (81/19 mol % After Purification)

(44) The ATRP initiator iv (FIG. 1) (70.5 mg), SPMAK (0.935 g) and HEMA (2.3 g) were dissolved in 60 mL of a methanol/water (8/1) mixture and degassed under argon for 15 min. Bpy (115.8 mg) and Cu(I)Br (53.9 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=227 000 g/mol; M.sub.w/M.sub.n=2.27.

Example 25

Synthesis of 18-Arm Star Hydroxyethyl Methacrylate/Sulfopropyl Methacrylate Potassium Copolymer (82.4/17.6 mol % After Purification)

(45) The ATRP initiator iii (FIG. 1) (75.3 mg), SPMAK (0.923 g) and HEMA (2.31 g) were dissolved in 60 mL of a methanol/water (8/1) mixture and degassed under argon for 15 min. Bpy (115.8 mg) and Cu(I)Br (53.9 mg) were then added under stirring at 20° C. After 24 h, the solution was exposed to air and the polymer was purified as reported in Example 5. M.sub.w=342 000 g/mol; M.sub.w/M.sub.n=2.28.

Example 26

Assessment of Polymer″Gliadin Binding

(46) The binding selectivity and affinity of gliadin toward the synthesized polymers was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS-PAGE) using a 15% (w/v) separating gel. In addition, the polymers were separately screened for their reactivity toward control proteins, namely bovine albumin and/or bovine casein. Binding studies were carried out at pH 1.2 and 6.8 using hydrochloric acid and phosphate buffers, respectively. Polymer (80 mg/L) and protein (40 mg/L) were mixed together at pHs 1.2 and 6.8 and incubated for 2 h. The solutions were then centrifuged at 15 000 g for 30 min in order to separate the insoluble complex from free protein that remained in solution. The supernatant was then analyzed by SDS-PAGE to measure the amount of free protein.

Example 27

Selectivity of Poly(HEMA-Co-SStNa) Binding to Gliadin

(47) The binding affinity of gliadin toward different linear poly(HEMA-co-SStNa) (synthesis reported in Examples 5 to 10 and 12-14) was assessed by SOS-PAGE as described in Example 26 and compared to that of albumin and casein (FIG. 3 showing Examples 5 to 10 and 12-13) at intestinal (6.8) and gastric (1.2) pHs. In general, the polymer exhibited greater affinity for gliadin compared to the control proteins at both pH values. It has to be pointed out that the complexation with casein was not studied at pH 1.2 due to the insolubility of this protein under acidic conditions. As shown by FIG. 3, complexation to gliadin could be modulated by the copolymer composition. FIG. 2 also shows selective binding on SOS-PAGE between gliadin and linear poly(HEMA-co-SStNa) (Example 10), whereas albumin remained free in solution upon incubating the copolymer with both proteins. The binding affinity of gliadin toward the linear poly(HEMA-co-SStNa) polymer of Example 14 was assessed by SDS-PAGE as described in Example 26 and compared to that of albumin. Results were as follows: complexation with albumin at pHs 1.2 and 6.8 was of 78.8% and 11.23%, respectively; complexation with gliadin at pHs 1.2 and 6.8 was of 100% and 71.3%, respectively.

Example 28

Selectivity of Poly(HEMA-co-SPMAK) Composition Binding to Gliadin

(48) The binding affinity of gliadin toward different linear poly(HEMA-co-SPMAK) (synthesis reported in Examples 18 to 22) was assessed by SDS-PAGE as described in Example 26 and compared to that of albumin (FIG. 4) at intestinal (6.8) and gastric pHs (1.2). Lesser binding to gliadin was observed when the SStNa monomer was replaced by SPMAK especially at pH 6.8 (FIG. 4). Optimal complexation to gliadin was achieved for SPMAK ratios ranging from 50 to 100 mol %.

Example 29

Effect of Copolymer Structure on Binding to Gliadin

(49) Five, eight and eighteen arms star poly(HEMA-co-SStNa) (Examples 15 to 17) and poly(HEMA-co-SPMAK) (Examples 23 to 25) were synthesized using initiators derived from glucose, sucrose and cyclodextrin, respectively. Their ability to bind gliadin was compared to their linear counterpart (Examples 9 and 18, respectively). The results are presented in FIGS. 5 and 6. For a fixed percentage of SStNa of about 30 mol %, the binding efficiency of eight arms star poly(HEMA-co-SStNa) was better than the linear or the other star copolymers (FIG. 5). At a fixed ratio of SPMAK of 17 mol %, no significant difference was observed in the binding of gliadin to linear or star poly(HEMA-co-SPMAK) (FIG. 6).

(50) Conclusions

(51) Linear and star-shaped random copolymers of HEMA and SStNa or SPMAK were shown to bind α-gliadin under pH conditions mimicking the gastrointestinal tract.

Example 30

Effect of Copolymer Molecular Weight on Binding to Gliadin

(52) Two different weights linear poly(HEMA-co-SStNa) (Examples 10 and 11) containing about 50% SStNa were tested for the binding of gliadin and albumin at both pHs 1.2 and 6.8. In this experience, each protein was tested separately. The results are presented in FIG. 9. The binding to gliadin and selectivity of binding was found to be influenced by the molecular weight of the polymer.

Example 31

Prevention of Enzymatic Degradation of Gliadin by a Copolymer

(53) Preparation of Peptic-Tryptic Digests of gliadin

(54) The stepwise enzymatic hydrolysis of α-gliadin was performed with pepsin (Sigma P0609; St Louis, Mo., USA) and trypsin (Sigma T1763), both attached to agarose as well as a-chymotrypsin from bovine pancreas (Sigma C4129). α-Gliadin (10 mg) was dissolved in 5 mL of hydrochloric acid buffer pH=1.2 (10 mM) and pepsin (38 U) was added. The mixture was magnetically stirred at 37° C. for 2 hours at which point the pH was adjusted to 6.8 with 0.1 mol/L NaOH and trypsin (0.75 U) as well as α-chymotrypsin (0.5 U) were added. The digest was centrifuged for 30 min at 20° C. and 6000 g. The gliadin peptides were thereafter collected in the supernatant and filtered through 0.2 μm GHP filters.

(55) The resulting peptic-tryptic-chymotryptic digest of gliadin was analyzed using a Waters™ high-performance liquid chromatography HPLC system equipped with a 1525 Binary pump, a 2487 dual wavelength absorbance detector, and a Breeze Chromatography Software™ (Waters, Midford, Mass.). Samples were eluted at 36° C. at a flow rate, detection wavelength, and injection volume of 1 MI/min, 215 nm and 50 μL, respectively. Trifluoroacetic acid was used as an ion pairing agent, and elution was performed with a linear gradient consisting of 100% buffer A to 100% buffer B spanning over 60 min. Buffer A consisted of 0.1% trifluoroacetic acid, 95% water, and 5% acetonitrile and buffer B consisted of 0.1% trifluoroactic acid, 5% water, and 95% acetonitrile. A portion of each sample supernatant was diluted into water and analysed on a C.sub.18 reversed phase column (Waters Novapack™ C18, 60 Å, 4 μm, 3.9×300 mm).

(56) Enzymatic Degradation of the Gliadin-Polymer Complex

(57) Poly(HEMA-co-SStNa) (Example 10) (4 g/L) and gliadin (2 g/L) were mixed together at pH 2 and incubated for 2 h. Then, the stepwise enzymatic degradation of gliadin-polymer complex was performed as described above. The effect of the polymeric binder on the degradation of gliadin was analysed using HPLC as described above (FIG. 7).

(58) Substantially less degradation products were detected when the gliadin was complexed to the polymer (FIG. 7).

Example 32

Effect of Polymer on Caco-2 Monolayer Integrity

(59) The effect of poly(HEMA-co-SStNa) (Example 10) on Caco-2 cell monolayer integrity was assessed and compared to that of PEG (35 kDa) and PVP (58 kDa) (FIG. 8). Cells were seeded onto 12-well Transwell® polycarbonate filters (Corning, Acton, Mass.) at a seeding density of 2.5×10.sup.5 cell/cm.sup.2. Caco-2 were grown in Dulbecco's modified essential medium (DMEM) supplemented with 10% (v/v) foetal bovine serum, non-essential amino acid solution (0.1 mM), Hepes buffer pH 7.4 (10 mM) and penicillin-streptomycin (eq. 100 U/mL and 100 μg/mL). Medium was refreshed every 72 h. Cells were cultured for 21-28 days at 37° C., 5% CO.sub.2 to form a differentiated monolayer prior to the experiments. Toxicity studies were performed in complete DMEM medium. Transepithelial electrical resistance (TEER) readings were taken at pre-determined time-points using a Millicell™ Electrical Resistance System (Millipore Corp. Bedford, Mass.) with a single electrode (World Precision Instruments, Sarasota, Fla.).

(60) In both the Poly(HEMA-co-SStNa) and control polymers (PVP, PEG), the TEER measured after 24 hours showed a reduction of 10% of the initial value (FIG. 8). These results indicate that Poly(HEMA-co-SStNa) do not seem to strongly perturb the integrity of the Caco-2 cell monolayer.

Example 33

In Vivo Testing of Effect of Polymeric Binder on Reduction of Toxicity of Gliadin and Gliadin Degradation Products

(61) The ability of the polymer to reduce the toxicity of gluten is evaluated in vivo by measuring the immune response of animals that have been sensitized to gluten or its degradation products. The immune response is measured in transgenic mice expressing HLA-DQ8 (24) following oral administration of gluten or its degradation products in the presence or absence of polymeric binder.

Example 34

Incorporation of Polymeric Binder in Food

(62) The polymeric binder may be incorporated into gluten-containing food directed to individuals affected by celiac disease. The polymeric binder in such food may then counteract the deleterious effects of the gluten contained in the food when it is swallowed. Without being so limited, such food includes ready-cooked dishes, cereals, baked goods such bread, pastry, pies, cakes, muffins, cookies etc. Such food may incorporate the polymeric binder in a concentration of 0.01% to 10% (w/w). The polymeric binder can also be incorporated into non gluten-containing food for consumption in a meal containing gluten-containing food. Without being so limited, such non gluten-containing food includes spreads such as cheese, jams, butter or any food that can be eaten on or with gluten-containing food.

(63) Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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