(2S)-Pterosin A和TM-1-1Ps在大鼠之體內代謝及藥物動力學研究及中藥方劑四逆散在迷你豬之體內代謝研究

Metabolism of (2S)-Pterosin A and Pharmacokinetic Study on TM-1-1Ps in Rat, and Metabolic Study on Sinisan, a Traditional Chinese Medicine Prescription, in Miniature Pig

李泳平(Yung-Ping Lee)

指導教授 ： 李水盛

國立臺灣大學/藥學專業學院/藥學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.10466

摘要

利用本實驗室已建立的分析技術模式：液相層析-質譜及液相層析-固相萃取-核磁共振儀，鑑定代謝物之結構，除能提升藥物後續深入研究之效率，也能探討藥物潛在之藥理或毒理作用。
第一部分為降血糖先導化合物(2S)-pterosin A在大鼠之體內代謝研究，使用胃管餵針給予(2S)-pterosin A，研究大鼠尿中及糞便藥物的代謝物。總共鑑定出19個代謝物，其中16個經高效液相層析-固相萃取-樣品轉移-核磁共振(HPLC-SPE-TT-NMR)串聯技術鑑定而得，7個代謝物經進一步分離決定其結構，有12個代謝物為新化合物。phase I代謝物於C-3, C-10, C-12, C-13或C-14位置進行氧化，接著在C-10或C-14進行脫羧反應(decarboxylation)及產生C-12/C-14或C-14/C-12內酯反應(lactonization)。phase II代謝物為原化合物或phase I代謝物之葡萄糖醛酸共軛化合物。主要的代謝物為(2S)-14-O-glucuronylpterosin A (M9), (2S)-2-hydroxymethylpterosin E (M14)和(±)-pterosin B (M19)，所得代謝物擁有類似的紫外光發光團，因此由高效液相層析分析取得原化合物之回歸線推算各代謝物在大鼠尿液及糞便中含量，指出約71%的原化合物以原形和代謝物形式經尿中排出。
第二部分為TM-1-1Ps系列藥物在大鼠之藥物動力學研究，探討由天然物半合成製備之系列化合物活性、代謝物及毒性的研究，由於具磷酸基的TM-1-1Ps相較於TM-1-1具甚佳之水溶解度，且TM-1-1DP具較佳的心臟保護作用並在有效劑量下亦無震顫之中樞神經作用，藉由TM-1-1DP之代謝研究證明其為TM-1-1之前驅藥。靜脈注射(60 mg/kg/ rat)結果顯示：TM-1-1DP之半衰期約15分鐘；在血中有兩葡萄糖醛酸共軛代謝物(TM-1-1 GlcUA1和TM-1-1 GlcUA2)生成；雖然在腦部紋狀體內檢測到TM-1-1DP (iv後0−30 min)及TM-1-1 (小於100 ng/mL)，但和全血中的濃度分別相差約1000及100倍，確認服用有效劑量的TM-1-1DP可避免TM-1-1產生的腦部副作用。
至於單磷酸基取代之TM-1-1MPa及TM-1-1MPb分別靜脈注射(50.8 mg/kg/ rat)結果顯示：TM-1-1MPa之半衰期約14分鐘；在血中有葡萄糖醛酸共軛代謝物(TM-1-1a GlcUA)生成；在腦部紋狀體檢測到TM-1-1MPa (iv後0−150 min)及持續之TM-1-1a 約100 ng/mL。TM-1-1MPb之半衰期約94分鐘；在血中有葡萄糖醛酸共軛代謝物(TM-1-1b GlcUA)生成；在腦部紋狀體檢測到持續之TM-1-1MPb (72−338 ng/mL)及TM-1-1b (iv後45−120 min)。
具磷酸基的TM-1-1Ps在大鼠體內會去除磷酸基，其中9號位置取代較2號位置取代安定，接者產生phase II葡萄糖醛酸共軛代謝物。在腦部紋狀體分佈的比例，具磷酸基的TM-1-1Ps明顯低於其去磷酸之代謝物(TM-1-1、TM-1-1a和TM-1-1b)，應可避免TM-1-1產生的震顫中樞神經副作用。
第三部分為中藥傳統方劑四逆散在迷你豬隻體內代謝研究，在尿中共鑑定出50個化合物(1–50)，包含10個未在服用四逆散之大鼠尿液發現的原形配醣體成份，其中36個化合物藉由HPLC-SPE-TT-NMR配合HPLC-HRESIMS串聯技術鑑定而得，之中有5個新化合物及9個蘭嶼小耳迷你豬隻內生性代謝物。大部分之phase I代謝物為原配醣體成分水解而得，phase II代謝物主要為葡萄糖醛共軛化合物，另有從芍藥苷及白芍苷水解而得之苯甲酸和從黃酮類化合物氧化斷裂之苯基丙酮酸類化合物，分別生成甘胺酸共軛之phase II代謝物。
綜合三部分之研究，運用HPLC-SPE-TT-NMR配合HPLC-HRESIMS串聯技術分析平台，提供更多藥物各代謝物之結構資訊，提升藥物代謝研究之效率，以利更深入地探討藥物潛在作用及藥物動力學性質，提高藥物開發之速度及成功率。

關鍵字

液相層析-核磁共振儀 ； 代謝研究 ； 磷酸化前驅藥 ； 四逆散 ； 迷你豬

並列摘要

The utilities of established LC-MS and LC-SPE-NMR techniques assisted the structural characterization of metabolites. The information might enhance the efficiency of following drug discovery and disclose potential bioactivities or toxicities of the drug.
The metabolic profile of the potent hypoglycemic agent, (2S)-pterosin A (P), in rat urine via intragastrical oral administration was investigated in the first part. In total, 19 metabolites (M1–M19) were identified. Among these, 16 metabolites were characterized by high-performance liquid chromatography solid-phase extraction-tube transfer-NMR, and seven metabolites were further isolated from the treated urine to enable further structural determination. Twelve of these are new compounds. The phase I metabolites of P were formed via various oxidations at positions C-3, C-10, C-12, C-13, or C-14 followed by decarboxylation of C-10 or C-14, and lactonization at C-12/C-14 or C-14/C-12. The phase II metabolites were glucuronide conjugates from the parent compound or phase I metabolites. The major metabolites were found to be (2S)-14-O-glucuronylpterosin A (M9), (2S)-2-hydroxymethylpterosin E (M14), and (±)-pterosin B (M19). Quantitative HPLCanalysis of metabolites, based on similar UV absorption and use of the regression equation of P, indicated that ~71% P was excreted as metabolites in rat urine.
The activity, metabolism, and toxicity of TM-1-1Ps, semi-synthesized from the natural product, were studied in the second part. TM-1-1DP was proved to increase dramatically the water solubility and abolish the CNS effect of TM-1, a drug candidate for the treatment of acute myocardial infarction. TM-1-1DP was proved to be the prodrug of TM-1-1 via metabolic study. To clarify its CNS effect, the presence of parent compound and its metabolites in the brain were examined via microdialysis and HPLC-Fluorescence techniques. After iv injection (60 mg/kg, rat), the following results were shown: T1/2 of TM-1-1DP was about 15 min; in striatum TM-1-1DP was detected during the intervals of 0−30 mins after injection; the concentration of TM-1-1 in striatum was below 100 ng/ml; the individual concentration, however, was about one thousandth and hundredth of aorta blood. This study supports that TM-1-1DP will not cause CNS effect, even at a dose 4000 times as the effective dose.
After respective iv injection of TM-1-1MPa and TM-1-1MPb (50.8 mg/kg, rat), the following results were shown: T1/2 of TM-1-1MPa was about 14 min; in striatum TM-1-1MPa was detected during the intervals of 0−150 mins after injection; in striatum TM-1-1a was detected in 4 hours after injection, and the concentration was about 100 ng/mL. T1/2 of TM-1-1MPb was about 94 min; in striatum TM-1-1MPb was detected in 4 hours after injection, and the concentration was between 72 and 338 ng/mL; in striatum TM-1-1b was detected during the intervals of 45−120 mins after injection.
The metabolites were formed in rat through dephosphorylation of TM-1-1Ps, followed by glucuronization to yield TM-1-1s glucuronide. 9-Phosphorylated TM-1-1s are found to be more stable than 2-phosphorylated ones. The ratios of TM-1-1Ps in blood to striatum are much higher than those of TM-1-1s. Therefore, TM-1-1Ps may prevent the CNS side effect resulted from the administration of TM-1-1.
The metabolic profile of the traditional Chinese medicine, Sinisan, in miniature pig urine via intragastric administration was investigated in the third part. In total, 50 compounds, including 10 unchanged parent glycosides which were not found from Sinisan’s metabolic profile in rats’ urine, were identified. Among these, 36 compounds were characterized by HPLC-SPE-TT-NMR coupled with HPLC-HRESIMS, five of which are new and nine are endogenous metabolites of miniature pig. Most of phase I and phase II metabolites are hydrolytic products of parent glycosides and glucuronide conjugates, respectively, the latter having been reported as sulfate conjugates while the experimental animal is rat. Benzoic acid, obtained from hydrolysis of albiflorin and paeoniflorin, and phenylpropenoic acids, obtained from oxidative cleavage of flavones, formed phase II glycine conjugates.
In summary, the application of HPLC-SPE-TT-NMR coupled with HPLC-HRESIMS provides more structural information of drug’s metabolites to facilitate the metabolic study of drugs. The suitable design to obtain the detail about potential activity and pharmacokinetic properties of drugs may increase the speed and successive opportunity of drug development.

並列關鍵字

LC-NMR ； metabolic study ； phosphorylated prodrug ； Sinisan ； miniature pig

參考文獻

(1) Steinmetz, K. L.; Spack, E. G. The basics of preclinical drug development for neurodegenerative disease indications. BMC Neurol 2009, 9 Suppl 1, S2.

(2) Fisher, S. J.; Swaan, P. W.; Eddington, N. D. The ethanol metabolite acetaldehyde increases paracellular drug permeability in vitro and oral bioavailability in vivo. J Pharmacol Exp Ther 2010, 332, 326-333.

(3) Chaurasia, C. S.; Muller, M.; Bashaw, E. D.; Benfeldt, E.; Bolinder, J.; Bullock, R.; Bungay, P. M.; DeLange, E. C.; Derendorf, H.; Elmquist, W. F.; Hammarlund-Udenaes, M.; Joukhadar, C.; Kellogg, D. L., Jr.; Lunte, C. E.; Nordstrom, C. H.; Rollema, H.; Sawchuk, R. J.; Cheung, B. W.; Shah, V. P.; Stahle, L.; Ungerstedt, U.; Welty, D. F.; Yeo, H. AAPS-FDA workshop white paper: microdialysis principles, application and regulatory perspectives. Pharm Res 2007, 24, 1014-1025.

(4) Toma-Dasu, I.; Uhrdin, J.; Lazzeroni, M.; Carvalho, S.; van Elmpt, W.; Lambin, P.; Dasu, A. Evaluating tumor response of non-small cell lung cancer patients with (18)f-fludeoxyglucose positron emission tomography: potential for treatment individualization. Int J Radiat Oncol Biol Phys 2015, 91, 376-384.

(5) Atzrodt, J.; Derdau, V. Selected scientific topics of the 11th International Isotope Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds. J Labelled Comp Radiopharm 2013, 56, 408-416.