Modification Of CDCA With Comb-like Copolymers And Their Effects On Dissolving Cholesterol Gallstone

Gallstones (GS) are common in the general population, which remain a main cause of serious morbidity and mortality. Cholesterol gallstone is more than 80% in all GS. Most of the cholelithiasis patient would like to accept non-surgical therapy, which is an important option especially for gallstone patients unsuitable for surgery because of age or coexisting disease, and for some patients who prefer not to undergo surgical procedures. Chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) are effective agents in certain circumstances for the dissolution of cholesterol gallstones. Unfortunately, stone dissolution by oral agents requires months to years of therapy. Furthermore, CDCA often causes diarrhea and aminotransferas elevations, and UDCA may cause stone calcification. To improve the effect of CDCA on dissolving cholesterol gallstone, the water-soluble copolymers as polycarboxylic acid of comb-like copolymers (PCC) and C₆₀-PCC were synthesized, which being used for modification of CDCA and strengthening effects of CDCA on dissolving the cholesterol gallstones by their entropy-repelled properties of comb-shaped side chains in buffers of phosphate (PBS) and in model bile.The design theory of polymer was mainly employed to synthesis the conjugate of PCC-CDCA and C₆₀-PCC-CDCA by C₆₀, polyoxyalkylene allylalkyldiether(PAO) and maleic anhydride(MAn), which were copolymerized with AIBN as initiator, and then PCC or C₆₀-PCC were reacted with CDCA. The structures of the copolymers and the conjugates of PCC-CDCA and C₆₀-PCC-CDCA were confirmed with IR, TLC and TEM. The dissolving effects and the dissolution kinetics mechanism of cholesterol gallstone was inverstigated in PBS and in model bile of different cholesterol saturated index (CSI) at pH7.4, including PCC-CDCA or C₆₀-PCC-CDCA.It was showed that the conjugates of PCC-CDCA and C₆₀-PCC-CDCA were synthesized by results of IR, TLC and TEM. The diameter of CMP30, CMP20, CMP4, CMO10, CMO7 and CMO4 as carriers was 180, 110, 120, 210, 190 and 190 nm, respectively. It was determined that conjugates of PCC-CDCA and C₆₀-PCC-CDCA were better than controlled group with the results of gallstone dissolution in PBS and in model bile. With the PCC-CDCAs, the ratio of dissolving gallstone of CDCA, MP40-CDCA, MP30-CDCA, MP20-CDCA, MP10-CDCA, MP4-CDCA, MOP10-CDCA, MOP7-CDCA, and MOP4-CDCA was 32.33%, 62.76%, 55.01%, 82.53%, 48.36%, 37.5%, 73.86%, 58.13%, and 45.1% at 7d in PBS at pH7.4, respectively. The dissolution rate of CDCA, MP40-CDCA, MP30-CDCA, MP20-CDCA, and MP10-CDCA was 5.33, 9.615, 6.868, 17.59, and 5.717×10-5mg.cm-2.s-1 in PBS at pH7.4, respectively. In model bile, the ratio of dissolving cholesterol gallstone of CDCA, MP4-CDCA, MP20-CDCA, MOP4-CDCA and MOP10-CDCA was (1) CSI=0.6: 29.08%, 46.63%, 74.5%, 53.6%, and 67.5%, (2) CSI=1.0: 12.2%, 15.77%, 21.7%, 16.9%, and 20.5%, (3) CSI=1.6: 6.69%, 8.17%, 21.6%, 11.7%, and 18.3% at 7d, respectively. The dissolution rate of CDCA, MP20-CDCA, and MP4-CDCA was (1) CSI=0.6: 5.34, 21.0, and 8.53×10-8mg.cm-2.s-1, (2) CSI=1.0: 4.63, 7.18, and 6.17×10-8mg.cm-2.s-1, (3) CSI=1.6: 1.57, 4.13, and 3.10×10-8 mg.cm-2.s-1 in model bile, respectively. With the C₆₀-PCC-CDCAs, the ratio of dissolving gallstone of CDCA, CMP30-CDCA, CMP20-CDCA, CMP4-CDCA, CMO10-CDCA, CMO7-CDCA, and CMO4-CDCA was 10.06%, 24.88%, 51.563%, 15.76%, 35.83%, 27.33%, and 19.31% at 7d in PBS at pH7.4, respectively. The dissolution rate of CDCA, CMP4-CDCA, CMP10-CDCA, CMP20-CDCA, CMP30-CDCA, and CMP40-CDCA was 2.27, 2.51, 2.66, 3.48, 2.92, and 3.28×10-5mg.cm-2.s-1 in PBS at pH7.4, respectively.These cojugates were novel water soluble copolymers of CDCA and C₆₀. With PCC-CDCAs, the optimal conditions were obtained as follows: the concentration of initiator, AIBN, 0.1% - 1.0%, the copolymerization temperature 80℃, the reaction time 4.5h, the dehydration time, the reagent of dehydration is toluene, 3.5h, the reaction time of CDCA and comb-like copolymers 1.5h. With C₆₀-PCC-CDCAs, the optimal conditions were obtained as follows: the concentration of initiator, AIBN, 0.1% - 1.0%, the copolymerization temperature 80℃, the concentration of C₆₀ 1%, the reaction time 4-7h, the dehydration time, the reagent of dehydration is toluene, 3.5h, the reaction time of CDCA and comb-like copolymers 1.5h.Cholesterol dissolving effects in all PCC-CDCAs and C₆₀-PCC-CDCAs were significantly larger than in CDCA (P<0.005) in PBS and in model bile. The abilities of dissolving cholesterol gallstones were affected by the length of side chain and the properties of hydrophilic-lipophilic of comb-like copolymers. The dissolution kinetics studies suggest that the interfacial resistance was the dominant rate-determining factor on dissolving cholesterol gallstones both in PBS and in model. The effects of dissolving cholesterol gallstones can be improved by increasing the steric interactive potential energy of side chains of PCC-CDCAs and C₆₀-PCC-CDCAs, and taking into account the properties of hydrophilic-lipophilic of them. The MP20-CDCA, MOP10-CDCA, CMP20-CDCA and CMO10-CDCA have the most excellent effect of dissolving and dispersing to the cholesterol gallstones in all products in PBS. The MP20-CDCA and MOP10-CDCA also have the most excellent effect of dissolving and dispersing to the cholesterol gallstones in all products in these three model bile.