| Total glucosides of paeony (TGP) is the active compound extracted from the dried roots of Paeonia lactiflora Pall. TGP is the first plant-derived anti-inflammatory and immunomodulatory drug that is used to treat rheumatoid disease. TGP is able to improve symptoms of rheumatoid patient without evident toxicity or side effects. However, its slow onset of effects seriously limits its clinical application. This limitation is attributed to poor absorption of Paeoniflorin (Pae) in the gastrointestinal tract when administered orally. Low lipophilicity of Pae is a major reason for its poor absorption as indicated by several hydrophilic hydroxyls in its molecular structure. Therefore, enhancing the lipophilicity of Pae becomes medically important. An innovative drug, benzenesulfonyl paeoniflorin (CP-25) was synthesized via aromatic acid esterification. Expectedly, the new compound showed improved anti-inflammatory and immunomodulatory activity. However, studies on plasma protein binding rate, metabolism and excretion of CP-25 have not been studied yet. Acetylation reaction can enhance lipophilicity, improve absorption and cell permeability. Moreover, acetylated derivatives have more beneficial effects than the parent compounds, and acetylation products are endogenous substances, which are relatively safe. And aliphatic acid esterification of Pae has not been synthesized. In this research, in order to improve the synthesis yield, orthogonal test was employed to optimize the synthesis technology. Plasma protein binding rate, excretion and metabolism of CP-25 were investigated. Upon synthesis, the pharmacokinetic processes in rats under intragastric administration (ig), anti-inflammatory and immunomodulatory activities of 6-AP will be preliminarily studied.Objective:To optimize the synthetic technological condition for CP-25 through the single factor experiment and orthogonal test, it may provide a blueprint for its industrial production. Also, to develop CP-25 as a new anti-inflammatory and immunomodulatory drug, and to carry out a systemic study on plasma protein binding rate, excretion and metabolism of CP-25.6'-acetylpaeoniflorin (6-AP) was synthesized via acetylation. Further, to study pharmacokinetic processes of 6-AP in vivo and to compare these pharmacokinetic parameters with those of Pae. Finally, anti-inflammatory and immunomodulatory activities of 6-AP will also be evaluated and compare the results with that of Pae.Methods:1. Pae and benzene sulfochloride were chosen as starting materials, pryidine was chosen as deacid reagent and 4-dimethylamiopryidine (DMAP) was chosen as catalyst. CP-25 was synthesized under water-free condition with chloroform as solvent. The effects of reaction conditions, such as molar ratio, amounts of catalyst, deacid reagent, solvent, reaction temperature and reaction time were investigated by single factor test. An orthogonal test was used to optimize the synthesis technology of CP-25.2. High performance liquid chromatography (HPLC) technique was used to establish an ultrafiltration method to evaluate the plasma protein binding rate of CP-25. And compare the difference between CP-25 and Pae, species, combinations of anti-inflammatory and immunomodulatory drugs in plasma protein binding rate.3. HPLC technique was used to establish quantitative analysis method to evaluate concentration of CP-25 in urine, feces and bile of rats in different time periods.4. UHPLC-ESI-MS/MS technique was employed to establish Information-Dependent Acquisition (IDA-UHPLC-ESI-MS/MS) method to detect the metabolites of CP-25 in plasma, urine, feces and bile of rats in different time periods.5. Pae and acetic anhydride were chosen as starting materials to synthesize 6-AP via acetylation, and the structure was identified by 1H-NMR and 13C-NMR. The pharmacokinetic processes between Pae and 6-AP in rats were compared under ig by HPLC. The inhibition of 6-AP and Pae on chronic inflammation and immune imbalance in mice with ACD were evaluated by ear swelling, spleen index, histopathology of ear tissue and spleen, proliferation of T lymphocyte and the secretion levels of interleukin 10(IL-10)and IL-17.Results:1. The results of single factor experiment showed that molar ratio, deacid reagent amount, solvent amount and reaction time are the major factors. The optimum reaction conditions obtained by orthogonal design method were as follows:molar ratio was 1:2, reaction time was 10 h, deacid reagent amount was 4 mL and solvent amount was 20 mL. The yield of CP-25 arose from 20% to 58%.2. (1) At the concentration of 20,40,80 ?g·mL-1, plasma protein binding rates of CP-25 with rat plasma were (93.79±0.31)%, (92.23±0.23)% and (90.66± 0.32)%, and plasma protein binding rates of Pae with rat plasma were (19.86±2.66)%?(19.95± 2.13)%? (19.90± 0.97)%. (2) At the concentration of 20,40,80 ?g·mL-1, plasma protein binding rates of CP-25 with dog plasma were (96.00± 0.19)%, (95.28± 0.41)% and (94.75± 0.47)%, and with human plasma were (94.87± 0.24)%, (93.62± 0.17)% and (93.23± 0.21)%. (3) In 40 ?g·mL-1 diclofenac sodium plus CP-25 (20,40 and 80 ?g·mL-1) groups, plasma protein binding rate of CP-25 was decreased by 7.90%,6.97% and 6.44% (P<0.05). In 2000 ?g·mL-1 diclofenac sodium plus CP-25 (20,40 and 80 ?g·mL-1) groups, plasma protein binding rate of CP-25 was decreased by 43.42%, 40.01%and 37.66%(P<0.05). In 40 ?g·mL-1 leflunomide plus CP-25 (40 and 80 ?g·mL-1) group, plasma protein binding rate of CP-25 was decreased by 0.37% and 0.87%(P>0.05). In 40 ?g·mL-1 leflunomide plus CP-25 (20 ?g·mL-1) group, plasma protein binding rate of CP-25 was decreased by 1.95%(P<0.05). In 400 ?g·mL-1 leflunomide plus CP-25 (20,40 and 80 ?g·mL-1) groups, plasma protein binding rate of CP-25 was decreased by 10.88%,9.17% and 5.47%(P<0.05). In 40 ?g·mL-1 celecoxib plus CP-25 (80 ?g·mL-1) group, plasma protein binding rate of CP-25 was decreased by 0.65%(P>0.05). In 40 ?g·mL-1celecoxib plus CP-25 (20 and 40 ?g·mL-1) group, plasma protein binding rate of CP-25 was decreased by 3.46% and 1.97% (P<0.05). In 400 ?g·mL-1 celecoxib plus CP-25 (20,40 and 80 ?g·mL-1) groups, plasma protein binding rate of CP-25 was decreased by 11.36%,10.01% and 7.21%(P<0.05).3. The accumulative excretion amount and cumulative urinary excretion ratio of CP-25 in male rat urine within 48 hours amounted to 22.1861±2.5725 ?g and 0.2175±0.0252%, and in female rat urine within 48 hours amounted to 13.8342±0.9274 ?g and 0.1356±0.0091%, respectively. The accumulative excretion amount and cumulative fecal excretion ratio of CP-25 in male rat feces within 96 hours amounted to 1923.2050±184.5339 ?g and 18.4039±1.7659%, and in female rat feces within 96 hours amounted to 1442.3421±276.7033 ?g and 14.0442±2.3535%, respectively. The accumulative excretion amount and cumulative biliary excretion ratio of CP-25 in male rat bile within 12 hours amounted to 317.8285±8.0204 ?g and 3.1894±0.0805%, and in female rat bile within 12 hours amounted to 713.3196±37.7496 ?g and 7.1583±0.3788%, respectively. There are differences between male and female in excretion.4. Five metabolites of CP-25 were discovered in plasma, urine, feces and bile of rats, named as M1 (monomethylated metabolite), M2 (debenzenesulfonylated metabolite), M3 (deormaldehyde metabolite), M4 (deglycosylated metabolite) and M5 (bimethylated metabolite), respectively. Unknown metabolites were also identified and speculated with IDA methods. M1, M2 and M3 were detected in plasma, M2 was detected in urine, M2, M4 and M5 were detected in feces, and M2 was detected in bile.5. The structure of 6-AP was confirmed by'H-NMR and 13C-NMR. The absorption processes of 6-AP by oral administration belong to first order kinetics. The absorption of 6-AP by ig was more than those of Pae. To compare with pharmacokinetic parameters of Pae, the t1/2, t1/2?p and MRT were prolonged obviously, while AUC and Cmax increased.6-AP could inhibit chronic inflammation in ears of mice with ACD, improve the histopathology of ear and spleen, decrease spleen index and abnormal proliferation of T lymphocytes, enhance IL-10 production and reduce IL-17 secretion from lymphocyte. The anti-inflammatory and immunoregulation effects of 6-AP was superior to those of Pae.Conclusion:1. The production process of CP-25 is simple, reliable, and the output of CP-25 is moderation, suitable for the requirement of pilot scale.2. The rat plasma protein binding rate of Pae is low, while CP-25 is high. Significant species differences were observed in the plasma protein binding rate among rats, dogs and human. Low dosage of leflunomide and celecoxib did not influence the plasma protein binding rate of CP-25. Diclofenac sodium, high dosage of leflunomide and celecoxib did influence the plasma protein binding rate of CP-25.3. There was significant difference in excretion of CP-25 between male and female in rat urine, feces and bile. By intragastric administration, the prototype drug of CP-25 was mainly excreted through fecal excretion.4. The major metabolic pathway of CP-25 was methylation, debenzenesulfonylation and deglycosylation. Pae is the main metabolite of CP-25 in vivo.5.6-AP is the product of Pae-6'O-acetate. The oral absorption of 6-AP was higher than that of Pae, and the elimination process of CP-25 was slower than that of Pae. The anti-inflammatory and immunoregulation effects of 6-AP were markedly higher than that of Pae. |
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