| 摘要: | 背景：本實驗室先前研究產出先導化合物 Aza-PBHA (AzP) ，可抑制癌症化療或放射線治療引發之黏膜潰瘍，並於倉鼠動物模型，證明具有明顯創傷癒合效果，但是AzP溶解度不佳 (130 ng/mL) 而影響活體口服之療效，阻礙藥物後續開發。
目的：本論文嘗試以助溶劑增加AzP之溶解度，以人工腸液與胃液評估AzP之安定性，並進行藥物動力學試驗，建立AzP 於大鼠之血中濃度變化，以評估AzP之生體可用率。
方法： (1) 建立分析AzP之HPLC條件，探求偵測及定量極限、決定線性範圍、準確與精確度並進行分析方法確效；(2) 運用臨床可用之助溶劑、界面活性劑等增加AzP溶解度；(4) 進行AzP人工腸液與胃液之安定性試驗，瞭解AzP之生物穩定性；(5) 進行AzP之藥物動力學實驗；在藥動學試驗時使用LC/MS/MS進行樣品分析 (委託世宬生物科技公司分析)。得到血中濃度後以非室性模式計算藥動學參數。
結果：(1) AzP 之血漿樣品HPLC 的分析條件為：使用Rp-18e (250 x 4.0, 5 μm) 管柱，移動相為 acetonitrile /ethanol /water /Trifluoroacetic acid /2-Propanol (80: 14.13: 2.25: 0.27: 1.35)，流速1 mL/min，偵測波長 237 nm，可得 LOD 1.95 μg/mL，LOQ 3.13 μg/mL，線性範圍為3.13 - 50 μg/mL，r2 = 0.9999；並進行同日間與異日間確效 (r2 = 0.9997-0.9999)；LC/MS/MS分析方法為偵測 parent ion 548.90 m/z；daughter ion 227.30 m/z；偵測極限為 1 ng/mL，線性範圍 1-500 ng/mL；(2) 利用五種有機溶劑、六種助溶劑，將 AzP的水溶解度 130 ng/mL 提升至7 mg/mL (53,846 倍)。(3) AzP在人工腸液與胃液4小時之回收率皆在 97.15% ~ 105.99% 間，顯示其在腸胃道中相對穩定。(5) AzP在大鼠進行靜脈注射(15 mg/kg, n = 5) 藥動學試驗，以非室性藥動學模式計算得動力學參數如下：AUCINF 40.14±17.91 hr x ng/mL，Cmax 39.05±27.68 ng/mL，CL 557,056.41±532,758.10 mL/hr/kg，T1/2 2.18 ± 0.89 hr；腹腔注射 (80 mg/kg, n = 6) 之非室性模式藥物動力學參數如下：AUCINF 294.89 ± 64.29 hr x ng/mL，Tmax 2.83 ± 0.26 ng/mL，Cmax 15.85 ± 4.57 ng/mL，CL 282.09 ± 59.85 L/hr，T1/2 13.31 ± 4.97 hr；計算得生體可用率76.83%。
結論：透過溶劑與助溶劑組合，AzP之溶解度提升53,846倍。AzP 於靜脈注射時半衰期短、清除率快，而於腹腔注射半衰期與平均滯留時間長，顯示 AzP 於腹腔注射具有緩釋作用且腹腔生體可用率高。口服投藥尚未偵測出可能原因為尚未找到最適化的口服配方或 AzP 自身溶解度過低。
Background: Aza-PBHA (AZP) is a lead compound discovered in our previous study, which exhibited profound in vivo efficacy in hamster oral mucositis model. The poor water solubility (130 ng/mL) may hinder GI tract absorption. Thus, improving the water solubility by formulation design is essential before AzP being programmed for further studies.
Purpose: This study aims to improve the physic-chemical properties of AzP in order to optimize the PD/PK profile. Studies included are: (1) establish analytical methods and validation; (2) using clinical acceptable excipients (solvents, surfactants, aqueous solubility enhancing agents) for solubility optimization；(3) biological stability study of AzP in artificial intestinal and gastric fluid；(5) investigate the bioavailability of AzP in rats.
Methods: HPLC and LC/MS/MS were used in this study to analyze AzP in biological samples. Non-compartmental model was used to calculate PK parameters in pharmacokinetic studies.
Results and Discussion: (1) The plasma sample of AzP was analyzed on a Rp-18e (250 x 4.0, 5 μm) column, eluted at a flow rate of 1 mL/min with a solvent system consisting of acetonitrile /ethanol /water /Trifluoroacetic acid /2-Propanol (80: 14.13: 2.25: 0.27: 1.35) and detected by photodiode array at wavelength of 237 nm with detection limit LOD 0.78 μg/mL; LOQ 3.13 μg/mL; and linearity range 3.13 - 50 μg/mL, r2 = 0.9999. This analytical method was validated with intra-day and inter-day validation and showed good robustness (r2 = 0.9998). LC/MS/MS was used to analyze biological sample in PK study which monitor parent ion 548.90 m/z, daughter ion 227.30 m/z, detection limit is 1 ng/mg with linearity range 1-500 ng/mL. (2) After testing with various solvent and co-solvent, the solubility of AzP was improved by 53,000 folds (from 130 ng/mL to 7 mg/mL). (3) AzP is fairly stable after 4 hours of incubation in artificial gastro and intestinal fluid with recovery between 97.15% to 105.99 %. (4) Preliminary pharmacokinetic study was conducted. However, the plasma concentration of AzP was below the detection limit by oral administration. The time-concentration profile of AzP upon intravenous administration (15 mg/kg, n = 5) was established with key PK parameters in non-compartmental model: AUCINF 40.14 ± 17.91 hr.ng/mL，Cmax：39.05 ± 27.68 ng/mL，CL 557,056.41 ± 532,758.10 mL/hr/kg，T1/2 2.18 ± 0.89 hr. By intraperitoneal administration (80 mg/kg, n = 6), key PK parameters in non-compartmental model: AUCINF 294.89 ± 64.29 hr x ng/mL，Tmax 2.83 ± 0.26 ng/mL，Cmax 15.85 ± 4.57 ng/mL，CL 282.09 ± 59.85 L/hr，T1/2 13.31 ± 4.97 hr。
Conclusion: The solubility of AzP in water-based vehicle was improved to 53,000 folds in this study. In i.v. administration, AzP showed short half-life and fast clearance. In i.p. administration, the half-life was greatly improved which suggest that AzP will exhibit slow-releasing profile after i.p. administration. The low systemic exposure after oral administration suggested further formulation study to improve AzP bioavailability. |
