[en] β-Cyclodextrin (β-CD) was monofunctionalized into its carboxylic derivative and then conjugated to the N-side of oxytocin (OT), a nonapeptide involved in human behavior and myometrium contraction. On isolated rat myometrium, this conjugate (β-CD-OT) partly preserves the contracting activity of OT (EC50 = 0.40 μM vs 1.7 nM). Moreover, the contraction induced frequency is also lowered by β-CD-OT. This novel hydrophilic targeted carrier could form a host–guest complex with prostaglandins and their derivatives used as labor inducers or with anticancer drugs used in cervix and endometrial cancer. This strategy can improve the solubility, the stability, and/or the biological activity of these drugs as well as reducing their side-effects.
Disciplines :
Pharmacy, pharmacology & toxicology
Author, co-author :
Bertolla, C; Université de Liège - ULiège > Département de pharmacie > Pharmacie galénique
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Mono-6-deoxy-6-(p-tosylsulfonyl)-β-cyclodextrin (2). In an E-flask equipped with a magnetic stirrer, sodium hydroxide (8.8 g; 220 mmol) was added to a suspension of β-CD (1, 25 g; 22 mmol) in distilled water. Then, 1-(p-toluenesulfonyl)imidazole) (5 g; 22.5 mmol) was added. After 1 h, hydrochloric acid, 0.1 N, was added dropwise to reach pH 5-6. The white formed precipitate was collected, washed with hot water (200 mL), with cold water (200 mL), with acetone (100 mL), and dried to give 9.23 g (yield = 33%) of the title compound 2. 1H NMR (400 MHz, DMSO-d6)δ 2.44 (s, 3H), 3.20-3.69 (overlap with D2O, 40 H), 4.17-4.22 (m, 1H), 4.33-4.40 (m, 2H), 4.45-4.49 (m, 2H), 4.53 (br s, 3H), 4.78 (br s, 2H), 4.85 (br s, 5H), 5.65-5.86 (m, 14H), 7.44 (d, 2H), 7.76 (d, 2H); m/z 1311 [M+Na]+. Anal. Calcd for C49H76O37S·3.5H2O: C, 43.23; H, 6.17. Found: C, 43.05; H, 5.94.
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Mono-6-deoxy-6-formyl-β-cyclodextrin (3). A microwave vial containing DMSO (10 mL) was filled with β-cyclodextrin monotosylate (2, 1.02 g; 0.78 mmol) and collidine (1 mL; 7.76 mmol). The vial was sealed with a teflon septum and irradiated at 2.450 GHz and 200 °C for 20 min (Biotage AB microwave oven, Uppsala). After heating, the vial was cooled, and its content added to acetone (200 mL). The formed precipitate was collected and dissolved in water (10 mL). This solution was added dropwise to ethanol (100 mL). The formed precipitate was collected, washed with ethanol, purified by preparative LC/MS (Econosphere-Alltech column, 250 cm × 22 mm; particle size 10 μm; mobile phase acetic acid 0.1%: CH3CN (80:20 v/v); flow rate: 5 mL min-1) to give 2.63 g (yield = 60%) of the titled compound 3. 1H NMR (400 MHz, DMSO-d6) (note: this contains both the aldehyde and covalent hydrate form of the compound) δ 3.26-3.81 (overlap with D2O, 40H), 4.16 (d, 1H), 4.38-4.45 (m, 6H), 4.79 (br s, 6H), 4.85-4.90 (d,1), 5.11 (t, 0.6H), 5.48 (d, 0.6H), 5.49 (d, 0.6H), 5.68-5.85 (m, 12H), 9.67 (s, 0.3); m/z 1133.1 [M+H]+, 1155.3 [M+Na]+. Anal. Calcd for C42H68O35·4H2O: C, 41.86; H, 6.31. Found: C, 42.06; H, 6.02.
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Mono-6-deoxy-6-carboxy-β-cyclodextrin (4). Bromine (4 mL; 0.90 mmol) was added to a solution of 6-deoxy-6-formyl-β-cyclodextrin (3, 1.02 g; 0.90 mmol) in 0.1 M phosphate buffer (10 mL; pH 6.0). The solution was stirred for 5 days at room temperature in the dark. Then, excess of bromine was extracted by diethyl ether (4 × 20 mL). The aqueous phase was added to acetone (600 mL), and the formed precipitate was collected and purified by preparative LC/MS according to the above described procedure to give 0.21 g (yield = 20%) of the title compound 4. 1H NMR (400 MHz, DMSO-d6) δ 3.37-3.74 (overlap with D2O, 40 H), 3.83 (d, 1H), 4.38-4.45 (m, 6H), 4.73-4.97 (m, 7H); 5.63-5.75 (m, 14H); m/z 1150.1 [M+H]+, 171.2 [M+Na]+. Anal. Calcd for C42H68O36·4H2O: C, 41.31; H, 6.23. Found: C, 41.55; H, 6.11.
Mono-6-deoxy-6-oxytocinyl-β-cyclodextrin (5). 6-Deoxy-6-carboxy-β-cyclodextrin (4, 0.52 g, 0.45 mmol) was dissolved in DMF (3 mL) and the solution cooled to 0 °C. Then, HOBt (88 mg, 0.54 mmol) in DMF (2 mL) and DCC (111 mg, 0.54 mmol) in DMF (2 mL) were added. The solution was stirred and the temperature maintained at 0-4 °C. After 1 h, oxytocin (0.54 mg, 0.54 mmol) dissolved in DMF (5 mL) was added to the reactive mixture, which was stirred for 48 h at room temperature. At the end of the reaction, 200 mL of acetone was added, the formed precipitate was collected, washed with acetone, dried, and purified by preparative LC/MS according to the above described procedure to give 0.21 g (yield = 30%) of the title compound 5. 1H NMR (DMSO-d6) δ 0.77-0.84 (m, 12H, OT), 2.29-2.50 (overlap with DMSO, 14H, OT), 3.08-3.93 (overlap with D2O, 41H (CD) + 11H (OT)), 4.38-4.45 (m, 6H), 4.53-4.70 (br m, 11H, OT), 4.73-4.97 (m, 7H, CD); 5.63-5.75 (m, 14H, CD), 5.91 (br s, 4H, OT), 7.10 (d, 2H, OT) 7.70 (d, 2H, OT), 8.46 (s, 8H, CONH); m/z 2138.1 [M+H]+, 1070.1 [M+2H]2+.
Female rats (Wistar, 200-250 g, Charles River Laboratories, Bruxelles, Belgium) are pretreated with β-estradiol benzoate (500 μg kg-1, ip, Sigma, Brussels, Belgium) 24 h before the experiment. Rats were killed with pentobarbital (80 mg kg-1, ip, Ceva Sante Animale, Brussels, Belgium) and myometrial tissue was removed and carefully trimmed of surrounding connective tissue. Uterine segments were placed longitudinally in a 20 mL tissue bath (EMKA Technologies, Paris, France) containing Krebs solution (mM: NaCl 118, KCl 5.4, CaCl2 2.5, MgCl2·6 H2O 1.5, NaHCO3 25, NaH2PO4 1.2, glucose 10; pH 7.4) continuously bubbled with a mixture of O2:CO2 (95:5%) at 37 °C. Each uterine segment was placed under optimum resting tension of 1 g and allowed to equilibrate for 1 h before the experiment. During this equilibrium period, the buffer was renewed every 15 min before exposing the uterine segments to the contractile agent (OT or β-CD-OT). When a stable tension was obtained, cumulative increasing concentrations of OT or β-CD-OT (ranging from 10-13 to 10-5 M) were added to the bath until tension and frequency are stable. Contractile response of myometrial tissue segments was recorded isometrically with a force-displacement transducer IT1 (EMKA Technologies, Paris, France). The EC50 value of each drug was assessed for at least three concentration-response curves and corresponded to the concentration which elicited 50% the maximal tension. The EC50 values were calculated by nonlinear regression analysis (GraphPad Prism software). The results are expressed as means ± SEM, n ≥ 3.
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Pymol software (v 1.0) was used to measure the distances from the crystal structure of oxytocin deposited with the RCSB Protein Data Bank; 1npo. doi:10.2210/pdb1npo/pdb.
