Caffeic acid 在家兔體內的藥物動態學及代謝的研究
中文摘要
Caffeic acid (CA) 屬於多酚類化合物，廣泛存於中藥、蔬果、茶及咖啡中，具有消 炎、保肝、抑制血小板凝集和抗腫瘤等藥理作用。但其藥物動態學及代謝途徑的 研究非常少且不是很詳細。因此，本研究的目的即以家兔為實驗動物，探討 CA 的藥物動態學，並運用 LC/MS/MS 研究 CA 的代謝途徑。靜脈注射不同劑量之 CA，其血漿中濃度經時變化，可以二室模式表示。投予劑量 5 mg/kg 所得之
beta-half life (11.1 +/- 1.6 分鐘) 很明顯地比投予劑量 10 mg/kg (14.3 +/- 2.9 分 鐘) 或 25 mg/kg (13.6 +/- 1.8 分鐘) 短，而投予劑量 5 mg/kg 所得之清除率 (0.0263 +/- 0.0025 l/kg/min) 亦比投予劑量 10 mg/kg (0.0200+/- 0.0054 l/kg/min) 或 25 mg/kg (0.0219+/-0.0050 l/kg/min) 大；顯示 CA 在投予劑量 10 與 25 mg/kg 範 圍內，其藥物動態學不受劑量的影響，但在較低的劑量下，其藥物動態學則呈 現 dose-dependent。此現象主要來自投予劑量 5 mg/kg 所得之 腎清除率 (CLr ， 0.0168 +/- 0.0029 l/kg/min) 與腎排除速率常數(kr，0.112+/- 0.013 (1/min)) 很明 顯地比投予劑量 10 mg/kg (CLr ，0.0120+/- 0.0035 l/kg(1/min)；kr，0.0789+/- 0.0089 (1/min)) 或 25 mg/kg (CLr ，0.0125+/- 0.0049 l/kg/min；kr，0.0668 +/- 0.0170 (1/min)) 大。k12/k21、k12/k 10 及 k21/k10 的比值近於 0.5，可知 CA 由 central compartment 排除的速率，比分佈到 peripheral compartment 快。在分別投 予劑量 5、10 與 25 mg/kg 後，CA 以原型由尿中排出的比率分別為 63.4 +/-
6.1、60.0+/- 6.4 及 55.4+/- 11.3 (%)，且在投藥後 2 小時內，大部分即由尿中排出。
口服投予家兔 CA 後，每隻家兔之血漿中濃度經時變化皆呈現雙峰或多峰之吸 收現象。依投予劑量 (5, 10 25 mg/kg)，absorption-half life 分別為 39.7+/-
11.4 、40.2+/- 16.3 及 54.0 +/- 29.9 (分鐘)；Tmax 分別為 33.3+/- 9.8、25.8+/-15.3 及 24.2+/- 13.2 (分鐘)；Cmax 分別為 0.810+/- 0.337、1.69 +/- 0.92 及 3.95+/- 2.02 (ug/ml)；生體可用率分別為 0.364+/- 0.052、0.379+/- 0.037 及 0.402+/- 0.087。由 CA 直接投於十二指腸或膽汁引流後口服投予 CA，家兔之血漿中濃度經時變化 仍呈現雙峰或多峰之吸收現象，及 CA 由膽汁排除的比例小於 0.1 (%) 的結果，
顯示 CA 口服投予家兔後，血漿中 CA 濃度經時變化皆呈現雙峰或多峰之吸收 現象的原因，應與 CA 本身的水溶解度有關，而與胃排空不規則及腸肝循環無 關。將 AUC 或 Cmax 對劑量作圖，皆可得一良好之線性相關 (Cmax = 0.00007 x dose + 0.0475, r = 0.741, p<0.05；AUC = 0.00840 x dose + 5.69, r = 0.909，
p<0.05)，顯示 CA 口服吸收呈現線性藥物動態學 (linear pharmacokinetics)。CA 以原型由尿中排出的比率平均為 23 (%)，且大部份的 CA 約在 8 小時內即由尿 中排出。腹腔注射投予 CA (10mg/kg)，血漿中濃度經時變化亦呈現雙峰之吸收 現象，生體可用率為 0.729 +/- 0.118，CA 以原型由尿中排出的比率為 43.8 +/- 8.3 (%)，而大部分的 CA 在 2 小時內即由尿中排出。胃腸道對 CA 的吸收比例為 0.530+/- 0.094；肝抽提率為 0.271 +/- 0.118，而胃腸道對 CA 的抽提率為
0.470+/- 0.094；另外，胃腸道對 CA 的抽提率對肝抽提率的比例約為 2.50，顯
示 CA 口服投予後大部份被胃腸道所清除，而非被肝臟清除。以酵素水解探討尿 液中 CA 與 glucuronic acid (CA-G) 及 sulfate (CA-S) 進行抱合反應的生成比例，
由靜脈注射 5 或 10 mg/kg 的 CA 後，所測得 CA 的比例，分別為 73.8 +/- 7.6 和 71.8 +/- 7.1 (%)，顯示 CA 約有近 30 (%) 為其它代謝物。而靜脈注射 10 mg/kg 所 得 CA-G 平均為 6.57 +/- 2.00 (%) 比 5 mg/kg 為 3.41+/- 0.80 (%) 多，且有顯著差 異，顯示 CA 代謝成 CA-G 具劑量依存的現象 (dose dependent)。另外，CA-G 的 生成比例與投予劑量間有一線性關係 (Y = 0.302 X — 0.0632, r = 0.718,
p<0.05)；靜脈注射 5 mg/kg 所得 CA-G (3.41+/- 0.80 %) 比 CA-S (7.06 +/- 2.08 %) 少，且有顯著差異；但靜脈注射 10 mg/kg 所得 CA-G (6.57 +/- 2.00 %) 比 CA-S (5.30 +/- 1.53 %) 多，不過並無顯著差異。顯示，sulfation 的酵素 (PAPS
transferase) 對 CA 具較高的親和性 (affinity)，但飽和性 (capacity) 較低；相反的，
glucuronidation 的酵素 (UDP glucuronyl-transferase) 對 CA 具較低的親和性，但 飽和性 (capacity) 較高。口服投予 5 或 10 mg/kg 的 CA 後，以酵素水解所測得 CA 的比例分別為 37.1 +/- 7.4 和 35.6 +/- 5.7 (%)，顯示 CA 口服投予至少約有近 35 (%) 的投予劑量為家兔所吸收。而口服投予 10 mg/kg 所得 CA-G 為 8.05 +/- 2.73 (%) 比 5 mg/kg 為 3.68 +/- 1.23 (%) 多，且有顯著差異。顯示口服投予與靜脈 注射一樣， CA 代謝成 CA-G 亦具劑量依存的現象 (dose dependent)，且 CA-G 的生成比例與投予劑量間亦有一線性關係存在 (Y = 0.507 X — 3.33, r = 0.860, p<0.05)。而口服投予 10 mg/kg 所得 CA-S 為 5.27 2.12 (%) 比 5 mg/kg 為 10.1+/- 3.6 (%) 少，且有顯著差異。分別比較同劑量下，CA-G 與 CA-S 的比例，
口服投予 5 mg/kg 所得 CA-G (3.68+/- 1.23 %) 比 CA-S (10.1 +/- 3.6 %) 少，且有 顯著差異；另外，口服投予 10 mg/kg 所得 CA-G (8.05 +/- 2.73 %) 比 CA-S (5.27+/- 2.12 %) 多，亦有顯著差異。如同靜脈注射的結果，顯示 CA 口服投予，
低劑量下，亦先進行 sulfation，故 glucuronidation 比例較低；但隨劑量增加，
sulfation 飽和，相對地，由 glucuronidation 取代。另外，口服投予後所得 CA-G 或 CA-S 與原型排除的相對比例皆比腹腔注射後所得的相對比例高約 3 倍，顯 示無論是進行 glucuronidation 或 sulfation，CA 在腸壁進行 conjugation 代謝比肝 臟容易。全血測得之的 CA 濃度對血漿 CA 濃度的比值 (blood-to-plasma ratio) 在
所測得的血漿CA 濃度範圍內維持一穩定的比值；而 RBC 內 CA 濃度對血漿
CA 濃度的比值 (RBC-to-plasma ratio) 也很穩定，同樣不受血漿中 CA 濃度變化 所影響。顯示 CA 會分佈至 RBC，但其分佈及與血球結合並不受濃度的影響 。 Cblood/Cplasma 所得之平均值為 0.710 +/- 0.058。以 LC/MS/MS 快速篩檢尿中的 代謝物，共有七個，分別為 isoferulic acid 、CA 進行 sulfation 代謝的產物 (CA-4- O-sulfate 及 CA-3-O-sulfate)，或 glucuronidation 代謝的產物 (CA-4-O-
glucuronide)。另外，亦可看到 CA 甲基化且又進行 sulfation 代謝 (CA-4-O- methyl, 3-O-sulfate)，或甲基化且又進行 glucuronidation 代謝的產物 (Glucuronyl 3-O-methyl-CA 及 Glucuronyl 4-O-methyl-CA)。而血漿中測得的代謝物共有五個，
分別為甲基化代謝物 isoferulic acid、ferulic acid、CA-4-O-sulfate、CA-3-O-sulfate
及 esculetin。膽汁中測到 3 個代謝物，皆為 CA 進行 sulfation 代謝的產物 (CA-4- O-sulfate、CA-3-O-sulfate 及 CA-4-O-methyl, 3-O-sulfate)。由 CA 行甲基化代謝後，
再進行二次代謝 glucuronidation 時，glucuronidation 的位置為 CA 的酸基 (Glucuronyl 3-O-methyl-CA 及 Glucuronyl 4-O-methyl-CA) 及 CA 進行
glucuronidation 的代謝位置為 4-hydroxy 時 (CA-4-O-glucuronide) 未見此代謝物 同時有甲基化代謝，加上 CA 行甲基化代謝又進行 sulfation 代謝的產物亦只有 一個 (CA-4-O-methyl, 3-O-sulfate)，顯示 CA glucuronidation 或 sulfation 與甲基 化代謝間具選擇性。另外，膽汁與血漿中所測得的代謝物皆為進行 sulfation 的代 謝物，無 glucuronidation 的代謝物，與大白鼠主要為 glucuronidation 不同；不 過，與人及狗血漿中測得其他藥物抱合代謝 (主要亦為 sulfation) 的結果相似，
顯示 CA 的代謝有物種的差異。
英文摘要
Pharmacokinetics and metabolism of caffeic acid (3,4-dihydrocinnamic acid, CA) were studied using rabbits as animal model. To the beginning, linear
pharmacokinetics of CA was studied. Three different doses (5, 10 and 25 mg/kg) were administered intravenously (IV) to six rabbits, respectively. The concentration-time profiles for CA could be fitted by a two compartment model for each dose. The results showed that total body clearance (CLtotal) and elimination rate constant from the central compartment (k10) after a 5 mg/kg dose were greater than those after the other two doses. Furthermore, the *-half life and mean residence time (MRT) after a 5 mg/kg dose were less than after the other doses. The AUC value increased linearly with dose within the range of 10 mg/kg to 25 mg/kg. Most of the unchanged caffeic acid was excreted in the urine within 2 hours. The percentages of unchanged caffeic acid excreted in the urine were 63.3, 60.0 and 55.4 (%) after doses of 5, 10 and 25 mg/kg, respectively, which was not significantly different. However, significant differences in the renal clearances (CLr) and renal excretion rate constant were observed with a 5 mg/kg dose compared to the other doses. On the other hand, nonrenal clearances (CLnr) and nonrenal excretion rate constants showed no dose related differences. The differences observed in CLtotal, k10, *-half life and MRT between a 5 mg/kg dose and the other doses could be explained on the basis of the differences in renal clearance and renal excretion rate constant. After oral
administration CA to rabbits, the concentration-time profiles of caffeic acid showed a double peak phenomenon. The pharmacokinetic parameters of the CLtotal, CLr, CLnr, and absorption phase half-life showed no significant differences for each dose after oral administration. These results indicated that CA showed linear
pharmacokinetics after oral administration in the dose range of 5and 25 mg/kg. The results also showed that the values of the AUC and Cmax increased linearly with a
dose in the range of 5 mg/kg to 25 mg/kg. The absolute bioavailability values of CA were 0.364, 0.379 and 0.402, and the percentages of unchanged CA excreted in the urine were 23.2, 22.7 and 22.0 (%) after doses of 5, 10 and 25 mg/kg, respectively.
There were no significant differences obtained for absolute bioavailability and also for percentages of unchanged CA excreted in the urine in these dose ranges. After intraperitoneal administration (IP, 10 mg/kg), the concentration-time profiles of CA also showed a double peak phenomenon. The absolute bioavailability value of CA were 0.729 and the percentages of unchanged CA excreted in the urine were 43.8 (%).
The calculated gastrointestinal (GI) and hepatic extraction were 0.470 and 0.271, respectively. Ratio of GI to hepatic extraction was 2.5 and showed GI eliminated CA more active than liver. Glucuronidation (CA-G) and sulfation (CA-S) of CA excreted in rabbit urine were determined enzymatically. The results showed that percentages of total CA (unchanged CA + CA-G + CA-S) excreted in the urine were 73.8 and 71.8 (%) after IV dosing (5 & 10 mg/kg), respectively. After oral dosing (5 & 10 mg/kg), percentages of total CA excreted in the urine was 37.1% and 35.6 (%), respectively.
The percentages of conjugation for CA (CA-conjugation) were 10.5 and 11.9 (%), CA-G were 3.41 and 6.57 (%), and CA-S were 7.06 and 5.30 (%) after IV
administration 5 and 10 mg/kg dose, respectively. After oral administration, the percentages of CA-conjugation were 13.8 and 13.3 (%), CA-G were 3.68 and 8.05 (%), and CA-S were 10.1 and 5.27 (%) for 5 and 10 mg/kg dose, respectively. There were no significant differences for percentages of total and conjugation CA excreted in the urine, for both doses neither IV nor oral. In addition, there were no significant differences for percentages of CA-conjugation excreted in the urine between
administration routes for each dose. However, the percentages of CA-G showed significant differences between doses, not only for IV administration but also for oral administration. There was a linear relationship between the percentages of CA-G and doses for both IV and oral administration. It indicated that glucuronidation of CA showed dose-dependent. The ratio of CA-G/CA-S increased with dose and showed significant differences between doses for both IV and oral administration. The results indicated that sulfation of CA showed high affinity and low capacity, and
glucuronidation of CA showed low affinity and high capacity, not only after IV but also after oral administration. The relative fraction of CA-conjugation/unchanged CA for oral administration was greater than that after IP administration and showed significant differences. The result indicated that the contribution of GI in CA conjugation was higher than that of liver. After IV administration of CA at dose 25 mg/kg, there was a blood to plasma concentration ratio for CA concentration at 0.710.
The blood to plasma concentration ratio was concentration independent and
erythrocyte to plasma concentration ratio, too. After intraduodenal administration or
oral administration following bile duct cannulation, the concentration-time profiles of CA showed a double peak phenomenon. The biliary excretion of CA was less than 0.1
%. These results indicated that the reasons for concentration-time profiles of CA showed a double peak phenomenon after oral administration were not resulting from gastric emptying variability and enterohepatic circulation. However, this was likely due to the poor water solubility of CA. The metabolic pathways of CA were
investigated with LC/MS/MS. It showed the sulfation of CA was predominant both in rabbit plasma and bile. Isoferulic acid and ferulic acid, methylation of CA, were found in plasma. In addition, a phase I oxidative metabolite, esculetin, was found in plasma but not in urine and bile. In urine, there were 7 metabolites identified. They included methylation of CA at 4-hydroxy group (isoferulic acid), sulfation at 3- or 4-hydroxy group of CA (CA-3-O-sulfate and CA-4-O-sulfate), methylation at 4-hydroxy group and sulfation at 3-hydroxy group (CA-4-O-methyl, 3-O-sulfate), glucuronidation at 4- hydroxy group (CA-4-O-glucuronide), and methylation either 3- or 4-hydroxy group and then glucuronidation at carboxylic acid group (Glucuronyl 3-O-methyl-CA and Glucuronyl 4-O-methyl-CA).
